Increased Chromatin Condensation Research Articles

Investigation of the efficacy of different cryoprotectants in the freezing of testicular tissue and epididymal sperm: Spermatological parameters, tissue viability and PARP-1 gene expression

The presented study covers testicular tissue and epididymal spermatozoa cryopreservation processes in bulls and aims to investigate the effects of these applications on spermatological parameters, cell viability in testicular tissue, and the expression of the PARP-1 gene, a DNA repair enzyme. Testes of 20 bulls over 2 years old, slaughtered in a slaughterhouse, were used in the study. After spermatological evaluations, the semen obtained from the cauda epididymis was frozen in liquid nitrogen vapor according to the straw method and stored in liquid nitrogen (−196 °C). Testicular tissue pieces obtained from the testicles were frozen by the slow freezing method in cryotubes in diluents containing Dimethylsulfoxide (DMSO) and Ethylene Glycol (EG) cryoprotectants and stored in liquid nitrogen (−196 °C). The total motility (TM) (85.89 ± 12.83 %), progressive motility (PM) (54.02 ± 15.77 %), and kinematic parameter values of fresh sperm were significantly higher compared to the TM (57.62 ± 13.13 %), PM (29.60 ± 10.76 %), and kinematic parameter values after thawing (P < 0.05). Significant decreases in plasma membrane integrity (PMI) and viability and an increase in chromatin condensation and morphological disorders in the head, middle part, and tail regions were observed in post-thaw semen samples (P < 0.05). When the effects of DMSO and EG on cell viability after thaw in frozen testicular tissue were evaluated, it was observed that the cell viability values of testicular tissues frozen with EG (45.70 ± 10.00) were statistically significantly lower than those frozen with DMSO (51.20 ± 7.70) (P < 0.05). When the effects of both cryoprotectants on gene expression in tissue and semen samples were examined, it was determined that gene expression increased on average 0.19 ± 0.27 times in the tissue samples in the DMSO group compared to fresh tissue samples and 0.17 ± 0.19 times in the tissue samples in the EG group. It was determined that gene expression levels increased by an average of 1.20 ± 1.08 times in post-thaw epididymal spermatozoa samples compared to fresh semen samples. The results show that cryopreservation can activate cellular repair mechanisms by stimulating PARP-1 gene expression and affect gene expression by activating specific pathways in tissues and cells.

Innate Immune Training Enhances the Reactivation of Latently Infected HIV-1 from Monocytic Cell lines

Abstract HIV virus can persist in a latent but activatable chronic state in resting CD4 +T-cells and macrophages that are more persistent, leading to the requirement of lifelong combination antiretroviral therapy (cART). The latency of HIV-1 in macrophage cells is associated with increased chromatin condensation induced by histone methylation at HIV-1 integration sites. Innate immune training of macrophages has been shown to relieve chromosome condensation through epigenetic rewiring. We hypothesized that training of macrophages could enhance the reactivation of HIV-1 by opening the chromatin to facilitate transcription in response to Latency Reversing Agents (LRAs). We used beta-glucan, Syk inhibitor and Rutaecarpine to train the THP89GFP cell line, which is an experimental model for latently infected HIV-1 monocytes. We found that these trained THP89GFP cells have higher transcription of HIV-1 specific genes in response to PMA stimulation. To check the epigenetic changes associated with training in the proviral HIV-1 DNA of THP89GFP, we performed a chromatin immunoprecipitation for the histone mark, H3K27Ac. Consistent with the higher induction of transcription of HIV-1 genes, we observed that multiple regions of the HIV-1 LTR had higher deposition of H3K27Ac in the trained macrophages. Our studies thus show that induction of trained innate immunity may be a viable approach to the reactivation of latently infected HIV-1. This finding supports the hypothesis that innate immune training stimuli could be developed as novel candidates for enhancing reactivation of latently infected HIV-1, which may facilitate elimination of macrophage reservoirs. This work was supported by the Intramural Research Program of NIAID, NIH This work was supported by the Intramural Research Program of NIAID, NIH and Office of AIDS research.

Cytotoxic n-Hexane Fraction of the Egyptian Pteris vittata Functions as Anti-breast Cancer Through Coordinated Actions on Apoptotic and Autophagic Pathways.

We investigated the possible anticancer mechanisms of Pteris vittata [PV] n-hexane extract on MCF-7 [breast cancer cell line]. Cultured cell lines were treated with various concentrations of this extract ± Baf-A1 [autophagic inhibitor]. Cells' viability, apoptotic markers [caspase-7, Bax, and Bcl-2], autophagic markers [light chain 3 [LC-3] and P62/SQSTM1]], and the tumor suppressor P53 and its mRNA were checked by their corresponding methods. Treated cell lines showed significant concentration and time-dependent reductions in cell viability in response to PV-n-hexane extract and also exhibited a concomitant induction of apoptosis [increased chromatin condensation, nuclear fragmentation, and pro-apoptotic Bax, and cleaved caspase-7 levels while decreased Bcl-2 levels] and autophagy [increased autophagosomes vacuoles, and LC3B II levels while decreased P62/SQSTM1 levels]. Moreover, PV-n-hexane extract-treated cells showed significant increases in the P53 and its mRNA levels. The addition of Baf-A1 reversed the PV-n-hexane extract autophagic effects and increased apoptotic cell percentage with a much increase in the cleaved caspase-7 and P53 protein and its mRNA levels. We concluded that the PV-n-hexane extract exhibits cytotoxic effects on the MCF-7 cell line with significant reductions in cell viability and concomitant autophagy and apoptosis induction. Inhibition of autophagy in the PV-treated MCF-7 cells enhances apoptosis via a p35-dependent pathway.

Effects of a Single Dose of X-Ray Irradiation on MMP-9 Expression and Morphology of the Cerebellum Cortex of Adult Rats.

Although radiation is a strategy widely used to inhibit cancer progression, which includes those of the neck and head, there are still few experimental reports on radiation effects in the cerebellum,particularly on the morphology of its cortex layers and on the Matrix metalloproteinases' (MMPs') expression, which, recently, seems to be involved in the progression of some mental disorders. Therefore, in the present study, we evaluated the morphology of the cerebellum close to the expression of MMP-9 from 4 up to 60days after a 15-Gy X-ray single dose of X-ray irradiation had been applied to the heads of healthy adult male rats. The cerebellum of the control and irradiated groups was submitted for an analysis of cell Purkinje count, nuclear perimeter, and chromatin density using morphometric estimatives obtained from the Feulgen histochemistry reaction. In addition, immunolocalization and estimative for MMP-9 expression were determined in the cerebellar cortex on days 4, 9, 14, 25, and 60 after the irradiation procedure. Results demonstrated that irradiation produced a significant reduction in the total number of Purkinje cells and a reduction in their nuclear perimeter, along with an increase in chromatin condensation and visible nuclear fragmentation, which was also detected in the granular layer. MMP-9 expression was significantly increased on 4, 9, and 14days, being detected around the Purkinje cells and in parallel fibres at the molecular layer. We conclude that the effects of a single dose of 15-Gy X-ray irradiation in the cerebellum were an increase in MMP-9 expression in the first 2weeks after irradiation, especially surrounding the Purkinje cells and in the molecular layers, with morphological changes in the Purkinje cell and granular cell layers, suggesting a continuous cell loss throughout the days evaluated after irradiation.

Diosmin in combination with naringenin enhances apoptosis in colon cancer cells.

Colon cancer is one of the most commonly diagnosed malignancies, which begins as a polyp and grows to become cancer. Diosmin (DS) and naringenin (NR) are naturally occurring flavonoids that exhibit various pharmacological activities. Although several studies have illustrated the effectiveness of these flavonoids as anti‑cancerous agents individually, the combinatorial impact of these compounds has not been explored. In the present study, the combined effect of DS and NR (DiNar) in colon cancer cell lines HCT116 and SW480 were assessed by targeting apoptosis and inflammatory pathways. The MTT assay was used to evaluate the effect of DiNar on cell proliferation, while Chou‑Talalay analysis was employed to determine the combination index of DS and NR. Moreover, flow cytometry was used to monitor cell cycle arrest and population study. The onset of apoptosis was assessed by DAPI staining, DNA fragmentation, and AnnexinV‑fluorescein isothiocyanate/propidium iodide (AnnexinV‑FITC/PI). The expression levels of apoptotic pathway markers, Bcl‑2, Bax, caspase3, caspase8, caspase9 and p53, and inflammatory markers, NF‑κβ, IKK‑α and IKK‑β, were assessed using western blotting and reverse transcription‑quantitative PCR. These results suggested that DiNar treatment acts synergistically and induces cytotoxicity with a concomitant increase in chromatin condensation, DNA fragmentation and cell cycle arrest in the G0/G1 phase. AnnexinV‑FITC/PI apoptosis assay also showed increased number of cells undergoing apoptosis in the DiNar treatment group. Furthermore, the expression of apoptosis and inflammatory markers was also more effectively regulated under the DiNar treatment. Thereby, these findings demonstrated that DiNar treatment could be a potential novel chemotherapeutic alternative in colon cancer.

Telmisartan Exerts Cytotoxicity in Scirrhous Gastric Cancer Cells by Inducing G0/G1 Cell Cycle Arrest.

This study aimed to assess the effects of telmisartan (TEL), a potential antitumor agent, and its mechanism of action in the regulation of apoptosis, autophagy, and cell cycle in scirrhous gastric cancer (SGC). The effect of TEL on the viability and chromatin condensation of OCUM-2M and OCUM-12 cells was assessed. Protein expression and the cell cycle were analysed using western blotting and flow cytometry, respectively. TEL inhibited cell proliferation in a dose-dependent manner and increased chromatin condensation and autophagy marker LC3-II levels in OCUM-12 cells. TEL also increased the proportion of cells in the G0/G1 phase transition. Apoptosis and autophagy are partially involved in the inhibitory effect of TEL on cell proliferation. Additionally, TEL caused G0/G1 cell cycle arrest. Therefore, TEL could be a promising treatment for SGC.

AIF3 splicing switch triggers neurodegeneration

BackgroundApoptosis-inducing factor (AIF), as a mitochondrial flavoprotein, plays a fundamental role in mitochondrial bioenergetics that is critical for cell survival and also mediates caspase-independent cell death once it is released from mitochondria and translocated to the nucleus under ischemic stroke or neurodegenerative diseases. Although alternative splicing regulation of AIF has been implicated, it remains unknown which AIF splicing isoform will be induced under pathological conditions and how it impacts mitochondrial functions and neurodegeneration in adult brain.MethodsAIF splicing induction in brain was determined by multiple approaches including 5′ RACE, Sanger sequencing, splicing-specific PCR assay and bottom-up proteomic analysis. The role of AIF splicing in mitochondria and neurodegeneration was determined by its biochemical properties, cell death analysis, morphological and functional alterations and animal behavior. Three animal models, including loss-of-function harlequin model, gain-of-function AIF3 knockin model and conditional inducible AIF splicing model established using either Cre-loxp recombination or CRISPR/Cas9 techniques, were applied to explore underlying mechanisms of AIF splicing-induced neurodegeneration.ResultsWe identified a nature splicing AIF isoform lacking exons 2 and 3 named as AIF3. AIF3 was undetectable under physiological conditions but its expression was increased in mouse and human postmortem brain after stroke. AIF3 splicing in mouse brain caused enlarged ventricles and severe neurodegeneration in the forebrain regions. These AIF3 splicing mice died 2–4 months after birth. AIF3 splicing-triggered neurodegeneration involves both mitochondrial dysfunction and AIF3 nuclear translocation. We showed that AIF3 inhibited NADH oxidase activity, ATP production, oxygen consumption, and mitochondrial biogenesis. In addition, expression of AIF3 significantly increased chromatin condensation and nuclear shrinkage leading to neuronal cell death. However, loss-of-AIF alone in harlequin or gain-of-AIF3 alone in AIF3 knockin mice did not cause robust neurodegeneration as that observed in AIF3 splicing mice.ConclusionsWe identified AIF3 as a disease-inducible isoform and established AIF3 splicing mouse model. The molecular mechanism underlying AIF3 splicing-induced neurodegeneration involves mitochondrial dysfunction and AIF3 nuclear translocation resulting from the synergistic effect of loss-of-AIF and gain-of-AIF3. Our study provides a valuable tool to understand the role of AIF3 splicing in brain and a potential therapeutic target to prevent/delay the progress of neurodegenerative diseases.

Human OVCA2 and its homolog FSH3-induced apoptosis in Saccharomyces cerevisiae.

Mammalian ovarian tumor suppressor candidate 2 (OVCA2) gene belongs to the family of serine hydrolase (FSH). This study aimed to elucidate the functional similarities of OVCA2 with its yeast homolog genes (FSH1, FSH2, and FSH3) regarding apoptosis. We found that the expression of OVCA2 in Saccharomyces cerevisiae increased production of reactive oxygen species (ROS), decreased cell growth, disturbed mitochondrial morphology, reduced membrane potential, increased chromatin condensation, and externalization of phosphatidylserine (PS) (annexin V/propidium iodide staining) indicating induced apoptotic cell death in yeast. We also showed that complementation of OVCA2 in fsh3Δ cells reduced cell growth and increased the apoptotic phenotypes. Collectively, our results suggest that complementation of human OVCA2 in fsh3Δ cells induced apoptosis in S. cerevisiae.

DNA damage and ovarian ultrastructural lesions induced by nickel oxide nano-particles in Blaps polycresta (Coleoptera: Tenebrionidae)

Nickel oxide nanoparticles (NiO-NPs) have extensively used in industrial and consumer products. The present study conducted to gain more knowledge about the safe use of NiO-NPs and also to understand their impact on the environment and biological systems. Herein, we examined the genotoxic and ultra-structural effects of a sublethal dose of NiO-NPs (0.03 mg/g) on the ovarian tissues of the ground beetle, Blaps polycresta. The mean diameter of NiO-NPs was 24.49 ± 3.88 nm, as obtained through transmission electron microscopy (TEM). In terms of DNA damage levels, the frequency of micronucleus (MN) formation was highly significant in the NiO-NPs treated group versus the controls. Besides, NiO-NPs treatment resulted in a significant increase in the tail length of comets. Further, electron microscopy revealed a progressive increase in chromatin condensation of the ovarian nurse and follicular cells, in addition to the accumulation of lysosomes and endo-lysosomes in their cytoplasm. In conclusion, NiO-NPs are capable of gaining access to the ovary of B. polycresta and causing DNA damage and a high degree of cellular toxicity in the ovarian cells. The present study highlights, for the first time, the adverse effects of these NPs to female gonads of insects and raised the concern of its genotoxic potential. It would be of interest to investigate NiO-NPs mediated intracellular ROS generation in future studies.

Phenolic Extract of Seagrass, Halophila ovalis Activates Intrinsic Pathway of Apoptosis in Human Breast Cancer (MCF-7) Cells

The marine ecosystem is considered as a treasure of numerous novel biologically active molecules. We investigated the anticancer potential of the phenolic extract of Halophila ovalis in breast cancer (MCF-7) cells and characterized the possible underlying molecular mechanism. The phenolic extract (5 µl) of H. ovalis effectively inhibited the growth of MCF-7 cells. The results of DAPI staining indicated that this phenolic extract potently induces apoptosis in MCF-7 cells which was observed by increased chromatin condensation in the treated cells. An increased expression of the active fragments of an executioner caspase, caspase 3 in phenolic extract-treated MCF-7 cells further confirms this apoptosis induction. In consequence, the loss of mitochondrial membrane potential was noticed in treated cells. The protein expression analyzes show decreased expression of the anti-apoptotic protein, Bcl-2, and DNA repair enzyme, PARP in treated cells indicating the probable molecular targets of apoptosis. Further, the phenolic extract of H. ovalis blocked the antioxidant defense system in MCF-7 cells by down-regulating the protein expression of a major transcription factor, Nrf-2 and regulatory antioxidant enzymes, SOD-2 and HO-1. These results show the presence of chemopreventive compound(s) in the phenolic extract, which offers a platform for future studies to identify the active principles.

Antitumor and apoptotic effects of quercetin on human melanoma cells involving JNK/P38 MAPK signaling activation

In this study, we investigated whether Quercetin has anti-cancer effects on A375SM and A375P human melanoma cells. Cell viability was assessed using an MTT assay. The proliferation of melanoma cells was measured by a wound-healing assay. Quercetin significantly decreased viability and proliferation of A375SM cells in a concentration-dependent manner. However, quercetin had no effect on A375P cells. DAPI staining showed increased chromatin condensation in a concentration-dependent manner, indicating apoptosis. Flow cytometric analysis indicated that quercetin suppressed the viability of A375SM cells by inducing apoptosis. Expression of quercetin-induced apoptosis proteins was investigated by Western blot analysis. Quercetin increased the expression of Bax, phospho-JNK, phospho-p38 and phospho-ERK1/2, cleaved poly-ADP ribose polymerase and decreased Bcl-2 in a concentration-dependent manner. We also investigated the in vivo tumor-growth inhibitory effect of quercetin. Quercetin (at 50 and 100 mg/kg) significantly decreased the A375SM tumor volume compared to the control group and increased apoptosis as assessed by the TUNEL assay. Immunohistochemistry staining revealed that the level of phosphor-JNK and phosphor-p38 increased in the quercetin-treated mice. These results indicate that quercetin inhibited the growth of A375SM melanoma cells through apoptosis and thus can be regarded as a new and effective chemo-preventive or therapeutic agent.

Suppression of EGFR/STAT3 activity by lupeol contributes to the induction of the apoptosis of human non‑small cell lung cancer cells.

The aim of this study was to investigate the underlying mechanisms responsible for the anticancer effects of lupeol on human non‑small cell lung cancer(NSCLC). MTT assay and Trypan blue exclusion assay were used to evaluate the cell viability. DAPI staining and flow cytometric analysis were used to detect apoptosis. Molecular docking and western blot analysis were performed to determine the target of lupeol. We found that lupeol suppressed the proliferation and colony formation of NSCLC cells in a dose‑dependent manner. In addition, lupeol increased chromatin condensation, poly(ADP‑ribose) polymerase (PARP) cleavage, sub‑G1 cell populations, and the proportion of AnnexinV‑positive cells, indicating that lupeol triggered the apoptosis of NSCLC cells. Notably, lupeol inhibited the phosphorylation of epithelial growth factor receptor(EGFR). A docking experiment revealed that lupeol directly bound to the tyrosine kinase domain of EGFR. We observed that the signal transducer and activator of transcription3 (STAT3), a downstream molecule of EGFR, was also dephosphorylated by lupeol. Lupeol suppressed the nuclear translocation and transcriptional activity of STAT3 and downregulated the expression of STAT3 target genes. The constitutive activation of STAT3 by STAT3 Y705D overexpression suppressed lupeol‑induced apoptosis, demonstrating that the inhibition of STAT3 activity contributed to the induction of apoptosis. The anticancer effects of lupeol were consistently observed in EGFR tyrosine kinase inhibitor (TKI)‑resistant H1975 cells (EGFR L858R/T790M). Taken together, the findings of this study suggest that lupeol may be used, not only for EGFRTKI‑naïve NSCLC, but also for advanced NSCLC with acquired resistance to EGFRTKIs.

Morphological and ultrastructural analysis of normal, injured and osteoarthritic human knee menisci.

The human meniscus plays a crucial role for transmission and distribution of load across the knee, as well as shock absorption, joint stability, lubrication, and congruity. The aim of this study was to compare the complex geometry, and unique ultrastructure and tissue composition of the meniscus in healthy (control) and pathological conditions to provide understanding of structural changes that could be helpful in the future design of targetted therapies and improvement of treatment indications. We analyzed meniscus samples collected from 3 healthy multi-organ donors (median age, 66 years), 5 patients with traumatic meniscal tear (median age, 41 years) and 3 patients undergoing total knee replacement (TKR) for end-stage osteoarthritis (OA) (median age, 72 years). We evaluated the extracellular matrix (ECM) organization, the appearance and distribution of areas of calcification, and modifications of cellular organization and structure by electron microscopy and histology. The ECM structure was similar in specimens from traumatic meniscus tears compared to those from patients with late-stage OA, showing disorganization of collagen fibers and increased proteoglycan content. Cells of healthy menisci showed mainly diffuse chromatin and well preserved organelles. Both in traumatic and in OA menisci, we observed increased chromatin condensation, organelle degeneration, and cytoplasmic vacuolization, a portion of which contained markers of autophagic vacuoles. Areas of calcification were also observed in both traumatic and OA menisci, as well as apoptotic- like features that were particularly prominent in traumatic meniscal tear samples. We conclude that meniscal tissue from patients with traumatic meniscal injury demonstrate pathological alterations characteristic of tissue from older patients undergoing TKR, suggesting that they have high susceptibility to develop OA.

Cell cycle acceleration and changes in essential nuclear functions induced by simulated microgravity in a synchronized Arabidopsis cell culture

Zero gravity is an environmental challenge unknown to organisms throughout evolution on Earth. Nevertheless, plants are sensitive to altered gravity, as exemplified by changes in meristematic cell proliferation and growth. We found that synchronized Arabidopsis-cultured cells exposed to simulated microgravity showed a shortened cell cycle, caused by a shorter G2/M phase and a slightly longer G1 phase. The analysis of selected marker genes and proteins by quantitative polymerase chain reaction and flow cytometry in synchronic G1 and G2 subpopulations indicated changes in gene expression of core cell cycle regulators and chromatin-modifying factors, confirming that microgravity induced misregulation of G2/M and G1/S checkpoints and chromatin remodelling. Changes in chromatin-based regulation included higher DNA methylation and lower histone acetylation, increased chromatin condensation, and overall depletion of nuclear transcription. Estimation of ribosome biogenesis rate using nucleolar parameters and selected nucleolar genes and proteins indicated reduced nucleolar activity under simulated microgravity, especially at G2/M. These results expand our knowledge of how meristematic cells are affected by real and simulated microgravity. Counteracting this cellular stress is necessary for plant culture in space exploration.

Polymeric chitosan-glycolipid nanocarriers for an effective delivery of marine carotenoid fucoxanthin for induction of apoptosis in human colon cancer cells (Caco-2 cells)

Fucoxanthin (FUCO), a marine carotenoid is photo-, and thermo-labile and poorly bioavailable due to its lipophilicity. Hence, we developed a chitosan (CS) + glycolipid (GL) nanogels (NGs) to increase cellular uptake and anticancer efficacy of FUCO (10 μM) in human colon cells (Caco-2). Effect of FUCO loaded in NGs with/with no GL was studied in comparison with micellar FUCO. Results showed that the cell viability was lower (p < 0.05) in NGs + GL (50.5%) compared to NGs (-GL) (66.5%) and the mixed micelles (72.5%) groups over 48 h exposure. An enhanced reactive oxygen species (ROS) generation was evident in NGs + GL (379.2%) group compared to NGs (−GL) and mixed micelles groups. Further, induction of apoptosis with an increased chromatin condensation and DNA fragmentation as evidenced in DAPI staining and DNA ladder assay were higher in NGs + GL group than other groups. Down-regulation of Bcl-2 (6.6 folds) was higher in NGs + GL group compared to NGs (−GL) (1.94 fold) and mixed micelles (1.19 fold) groups. Higher Bax up-regulation in NGs + GL compared to other groups supports the Bcl-2 down regulation. Mitochondrial membrane polarisation (ΔΨm) was higher in NGs + GL group (2.46 fold) compared to NGs (−GL) (1.91 fold) and mixed micelles (1.26 fold) groups. The cellular FUCO uptake illustrated a positive correlation between its level (pmol/106 cells) in NGs + GL (758.3) and enhanced caspase-3 activity (25.8 folds). This could be the reason for an increased apoptotic activity in NGs + GL group than other groups. Results demonstrate that delivery of FUCO in NGs + GL carrier aids cellular uptake and chemotherapeutic potential of FUCO. Results further demonstrate, for the first time, higher anti-cancer activity of FUCO loaded in NGs + GL and the effect was through ROS generation via a caspase dependent mechanism in Caco-2 cells.

Apoptosis and antimigration induction in human skin cancer cells by rhodomyrtone.

Rhodomyrtone is a bioactive compound extracted from Rhodomyrtus tomentosa leaves. It has been used as a traditional herb medicine for many years. Rhodomyrtone exhibits antibacterial activity, anti-inflammatory and antioxidant activities. However, the anticancer activity of rhodomyrtone has not been previously reported. The present study investigated the anticancer effect of rhomyrtone on human epidermoid carcinoma A431 cells. The cytotoxic and antiproliferative effects of rhodomyrtone on A431 cells were investigated by an MTT assay. Cell morphological alterations and apoptotic cells were observed with Hoechst 33342 staining following rhodomyrtone treatment. Flow cytometry and western blotting were performed to detect cell cycle and apoptosis induction. The results demonstrated that rhodomyrtone inhibited proliferation of A431 cells in a dose-dependent manner with IC50 value of 8.04±0.11 µg/ml. The results also indicated that rhodomyrtone increased chromatin condensation, nuclear fragmentation and apoptotic bodies in treated A431 cells in a time-dependent manner. Apoptosis was also induced through the activation of caspase-7 and poly (ADP-Ribose) polymerase cleavage. Flow cytometry analysis revealed that rhodomyrtone induced cell cycle arrest at the G1 phase. Notably, the non-toxic concentration of rhodomyrtone markedly inhibited A431 cell migration in a dose- and time-dependent manner. These finding suggested that rhodomyrtone may be used as an anticancer agent for human skin cancer.

Dehydrocostus lactone induces prominent apoptosis in kidney distal tubular epithelial cells and interstitial fibroblasts along with cell cycle arrest in ovarian epithelial cells

Dehydrocostus lactone (DHCL), a sesquiterpene lactone is well-known for its antiulcer, anti-hepatotoxic and anticancer activity. However, the studies concerning the safety/toxicity potential of DHCL toward the cells of normal origin remain unclear. The present study is aimed at investigating the toxicity potential of DHCL in renal distal tubular and interstitial fibroblast cell lines (MDCK and NRK-49F cells, respectively), and also in ovarian epithelial cell line (CHO cells). The MTT assay has predicted potential cytotoxic activity of DHCL against the cell line types with IC50 values of 0.99, 2.1 and 5.15 μM, respectively. The prominent dose-dependent (IC30,50 & 70) increase in the percentage of cells at subG1 phase in all the cell lines revealed apoptosis induction, further establishing the cytotoxic effect of DHCL. The DHCL exposure (4 h) revealed the induction of ROS in both renal cell lines, which is responsible for apoptosis induction. The NRK-49F cells displayed dose-wise (IC30-70) increase in chromatin condensation and membranous phosphatidylserine translocation further confirming apoptotic cell death. Also, their increase in BAX/Bcl-2 ratio, mitochondrial membrane permeability and caspase-3/7 activity establishes mitochondrial mediated apoptosis. In case of CHO cells, the higher percentage of cells at G2/M phase and expression of Cyclin B1 at lower concentration of DHCL (≤IC30), indicate mitotic arrest. The incidence of chromatid gaps and negligible micronuclei formation in treated cells (IC10–30) suggest that sub-lethal concentrations of DHCL exposure causes mitotic arrest in response to the damages by steady expression of Cyclin B1. Under in vitro condition, the study of DHCL’s potential cytotoxic effect on both kidney cells and ovarian epithelial cells indicated the possibility of adverse effects on normal healthy cells as well. Hence, the study recommends in-depth investigations on DHCL usage concerning its safety in therapeutic applications.

Salvia officinalis Induces Apoptosis in Mammary Carcinoma Cells Through Alteration of Bax to Bcl-2 Ratio

Salvia officinalis L. (sage) is one of the herbal plants frequently used in traditional medicine. Anti carcinogenic property of sage extract and the fact that it does not show toxic effect on normal cells, make this extract a proper candidate for cancer therapy. In order to assess the molecular mechanism underlying sage anticancer properties, the effect of sage hydro alcoholic extract was investigated on mouse mammary carcinoma cell line (4T1). Cultured cells were treated with different concentrations (0–1000 μg/mL) of Salvia officinalis extract for 24 h. Decrease in cell viability and increase in chromatin condensation were observed in a dose-dependent manner following treatment with sage extract. Most cultured cells were in the early stages of apoptosis in 100 μg/mL and in the late stages of apoptosis in 500 μg/mL of the extract. While Bax expression was increased significantly at 500 μg/mL of sage extract, no significant change was observed in the levels of Bcl2 expression in different concentrations of the extract compared to non-treated control. Therefore, Salvia officinalis hydro alcoholic extract shows cytotoxic and proapoptotic properties on mouse mammary carcinoma cells. Moreover, it seems that the induction of apoptosis in those cells is through increased in the ratio of Bax/Bcl2 as an apoptotic index.

Chetomin induces apoptosis in human triple-negative breast cancer cells by promoting calcium overload and mitochondrial dysfunction

Human triple-negative breast cancer (TNBC) is poorly diagnosed and unresponsive to conventional hormone therapy. Chetomin (CHET), a fungal metabolite synthesized by Chaetomium cochliodes, has been reported as a promising anticancer and antiangiogenic agent but the complete molecular mechanism of its anticancer potential remains to be elucidated. In our study, we explored the anti-neoplastic action of CHET on TNBC cells. Cytotoxicity studies were performed in human TNBC cells viz. MDA-MB-231 and MDA-MB-468 cells by Sulforhodamine B assay. It exhibited antiproliferative response and induced apoptosis in both the cell types. Cell cycle analysis revealed that it increases the sub G0/G1 phase cell population. Modulation of mitochondrial membrane potential, activation of caspase 3/7 and a remarkable increase in the expression of cleaved PARP and increased chromatin condensation was observed after CHET treatment in MDA-MB-231 and MDA-MB-468 cells. Additionally, an elevated level of intracellular Ca2+ played an important role in CHET mediated cell death response. Calcium overload in mitochondria led to release of cytochrome c which in turn triggered caspase-3 mediated cell death. Inhibition of calcium signalling using BAPTA-AM reduced apoptosis confirming the involvement of calcium signalling in CHET induced cell death. Chetomin also inhibited PI3K/mTOR cell survival pathway in human TNBC cells. The overall findings suggest that Chetomin inhibited the growth of human TNBC cells by caspase-dependent apoptosis and modulation of PI3K/mTOR signalling and could be used as a novel chemotherapeutic agent for the treatment of human TNBC in future.

The Natural Antiangiogenic Compound AD0157 Induces Caspase-Dependent Apoptosis in Human Myeloid Leukemia Cells.

Evasion of apoptosis is a hallmark of cancer especially relevant in the development and the appearance of leukemia drug resistance mechanisms. The development of new drugs that could trigger apoptosis in aggressive hematological malignancies, such as AML and CML, may be considered a promising antileukemic strategy. AD0157, a natural marine pyrrolidinedione, has already been described as a compound that inhibits angiogenesis by induction of apoptosis in endothelial cells. The crucial role played by defects in the apoptosis pathways in the pathogenesis, progression and response to conventional therapies of several forms of leukemia, moved us to analyze the effect of this compound on the growth and death of leukemia cells. In this work, human myeloid leukemia cells (HL60, U937 and KU812F) were treated with AD0157 ranging from 1 to 10 μM and an experimental battery was applied to evaluate its apoptogenic potential. We report here that AD0157 was highly effective to inhibit cell growth by promotion of apoptosis in human myeloid leukemia cells, and provide evidence of its mechanisms of action. The apoptogenic activity of AD0157 on leukemia cells was verified by an increased chromatin condensation and DNA fragmentation, and confirmed by an augmentation in the apoptotic subG1 population, translocation of the membrane phosphatidylserine from the inner face of the plasma membrane to the cell surface and by cleavage of the apoptosis substrates PARP and lamin-A. In addition, AD0157 in the low micromolar range significantly enhanced the activities of the initiator caspases-8 and -9, and the effector caspases-3/-7 in a dose-dependent manner. Results presented here throw light on the apoptogenic mechanism of action of AD0157, mediated through caspase-dependent cascades, with an especially relevant role played by mitochondria. Altogether, these results suggest the therapeutic potential of this compound for the treatment of human myeloid leukemia.