Apoptotic Population Research Articles

Autofluorescence imaging identifies tumor cell-cycle status on a single-cell level.

The goal of this study is to validate fluorescence intensity and lifetime imaging of metabolic co-enzymes NAD(P)H and FAD (optical metabolic imaging, or OMI) as a method to quantify cell-cycle status of tumor cells. Heterogeneity in tumor cell-cycle status (e. g. proliferation, quiescence, apoptosis) increases drug resistance and tumor recurrence. Cell-cycle status is closely linked to cellular metabolism. Thus, this study applies cell-level metabolic imaging to distinguish proliferating, quiescent, and apoptotic populations. Two-photon microscopy and time-correlated single photon counting are used to measure optical redox ratio (NAD(P)H fluorescence intensity divided by FAD intensity), NAD(P)H and FAD fluorescence lifetime parameters. Redox ratio, NAD(P)H and FAD lifetime parameters alone exhibit significant differences (p<0.05) between population means. To improve separation between populations, linear combination models derived from partial least squares - discriminant analysis (PLS-DA) are used to exploit all measurements together. Leave-one-out cross validation of the model yielded high classification accuracies (92.4 and 90.1 % for two and three populations, respectively). OMI and PLS-DA also identifies each sub-population within heterogeneous samples. These results establish single-cell analysis with OMI and PLS-DA as a label-free method to distinguish cell-cycle status within intact samples. This approach could be used to incorporate cell-level tumor heterogeneity in cancer drug development.

Increased Fas ligand expression of peripheral B-1 cells correlated with CD4+ T-cell apoptosis in filarial-infected patients.

Cellular hyporesponsiveness observed during helminth infections is attributed to factors such as antigen-presenting cells (APC) dysfunction, increased interleukin-10(IL-10), regulatory T cells and induction of CD4+ T (Th)-cell apoptosis. Increased Fas ligand (FasL) expression on the surface of B-1 cells and induction of apoptosis of Th cells by FasL-expressing B-1 cells due to helminth infection were demonstrated in murine model of helminth infection where as profile of FasL expression, Th-cell apoptosis and correlation between these two populations of cells in clinical filariasis remain unknown. In this study, we have scored the profile of apoptotic Th-cell population and FasL-expressing B-1 cells in different clinical categories of filariasis. The peripheral apoptotic T-helper cells were significantly increased in filarial patients compared to endemic controls. Expression of FasL on the surface of peripheral B-1 cells increased in filarial patients and positively correlated with peripheral apoptotic T-helper cells indicating FasL-expressing B-1 cells may be one of the important mediators of Th-cell apoptosis and immune anergy during filarial pathology.

Guggulsterone sensitized drug-resistant human hepatocarcinoma cells to doxorubicin through a Cox-2/P-gp dependent pathway

Previous researches indicated that cyclooxygenase-2 (Cox-2) might be involved in P-glycoprotein (P-gp)-mediated multidrug resistance in hepatocellular carcinoma cells. Doxorubicin-resistant hepatocellular carcinoma PLC/PRF/5 cells (PLC/PRF/5R) and HepG2 (HepG2R) cells were developed in the present study. The modulatory effect of guggulsterone on Cox-2 and P-gp in PLC/PRF/5R and HepG2R cells was investigated. Cells proliferation, Cox-2 and P-gp expression, and prostaglandin E2 release were examined using MTT, flow cytometry, western blot and ELISA assays. Small interfering RNA (siRNA) targeted against Cox-2 and multidrug resistance protein (Mdr-1) was used to regulate the expression of Cox-2 and P-gp. The results showed that co-administration of guggulsterone resulted in a significant increase in chemo-sensitivity of PLC/PRF/5R cells to doxorubicin, as compared with doxorubicin treatment alone. When doxorubicin (10µM) was combined with guggulsterone (50µM), the mean apoptotic population of PLC/PRF/5R cells was 20.16%. It was increased by 1.5 times, as compared with doxorubicin (10µM) treatment alone. Furthermore, guggulsterone had significantly inhibitory effect on the levels of Cox-2, P-gp and prostaglandin E2. However, guggulsterone did not show significantly inhibitory effect on the expression of prostaglandin E receptors. In addition, Cox-2 siRNA simultaneously reduced the expression of Cox-2 and P-gp in PLC/PRF/5R cells. Mdr-1 siRNA had no influence on Cox-2, but inhibited P-gp expression. The present study suggested that guggulsterone might enhance the cytotoxic effect of doxorubicin to PLC/PRF/5R cells through a Cox-2/P-gp dependent pathway.

Apigenin overcomes drug resistance by blocking the signal transducer and activator of transcription 3 signaling in breast cancer cells.

Drug resistance in chemotherapy is a serious obstacle for the successful treatment of cancer. Drug resistance is caused by various factors, including the overexpression of P-glycoprotein (P-gp, MDR1). The development of new, useful compounds that overcome drug resistance is urgent. Apigenin, a dietary flavonoid, has been reported as an anticancer drug in vivo and in vitro. In the present study, we investigated whether apigenin is able to reverse drug resistance using adriamycin-resistant breast cancer cells (MCF-7/ADR). In our experiments, apigenin significantly decreased cell growth and colony formation in MCF-7/ADR cells and parental MCF-7 cells. This growth inhibition was related to the accumulation of cells in the sub-G0/G1 apoptotic population and an increase in the number of apoptotic cells. Apigenin reduced the mRNA expression of multidrug resistance 1 (MDR1) and multidrug resistance-associated proteins (MRPs) in MCF-7/ADR cells. Apigenin also downregulated the expression of P-gp. Apigenin reversed drug efflux from MCF-7/ADR cells, resulting in rhodamine 123 (Rho123) accumulation. Inhibition of drug resistance by apigenin is related to the suppression of the signal transducer and activator of transcription 3 (STAT3) signaling pathway. Apigenin decreased STAT3 activation (p-STAT3) and its nuclear translocation and inhibited the secretion of VEGF and MMP-9, which are STAT3 target genes. A STAT3 inhibitor, JAK inhibitor I and an HIF-1α inhibitor decreased cell growth in MCF-7 and MCF-7/ADR cells. Taken together, these results demonstrate that apigenin can overcome drug resistance.

GATA2 Inhibition Sensitizes Acute Myeloid Leukemia Cells to Chemotherapy.

Drug resistance constitutes one of the main obstacles for clinical recovery of acute myeloid leukemia (AML) patients. Therefore, the treatment of AML requires new strategies, such as adding a third drug. To address whether GATA2 could act as a regulator of chemotherapy resistance in human leukemia cells, we observed KG1a cells and clinical patients’ AML cells with a classic drug (Cerubidine) and Gefitinib. After utilizing chemotherapy, the expression of GATA2 and its target genes (EVI, SCL and WT1) in surviving AML cells and KG1a cells were significantly enhanced to double and quadrupled compared to its original level respectively. Furthermore, with continuous chemotherapeutics, AML cells with GATA2 knockdown or treated with GATA2 inhibitor (K1747) almost eliminated with dramatically reduced expression of WT1, SCL, EVI, and significantly increased apoptotic population. Therefore, we propose that reducing GATA2 expression or inhibition of its transcription activity can relieve the drug resistance of acute myeloid leukemia cells and it would be helpful for eliminating the leukemia cells in patients.

The selective cytotoxicity of DSF-Cu attributes to the biomechanical properties and cytoskeleton rearrangements in the normal and cancerous nasopharyngeal epithelial cells

Cancer initiation and progression follow complex changes of cellular architecture and biomechanical property. Cancer cells with more submissive (or “softer”) than their healthy counterparts attributed to the reorganization of the complex cytoskeleton structure, may be considered as a potential anti-tumor therapeutic target. In this study, atomic force microscopy (AFM) was carried out to detect the topographical and biophysical changes of nasopharyngeal carcinoma CNE-2Z cells and normal nasopharyngeal epithelial cells NP69-SV40T by treating the Disulfiram chelated with Cu2+ (DSF-Cu). DSF-Cu induced the apoptotic population, ROS production and decreased the NF-κB-p65 expression of CNE-2Z cells, which was much higher than those of NP69-SV40T cells. DSF-Cu caused the obvious changes of cell morphology and membrane ultrastructure in CNE-2Z cells. The roughness decreased and stiffness increased significantly in CNE-2Z cells, which correlated with the rearrangement of intracellular F-actin, FLNa and α-tubulin structures in CNE-2Z cells. And the adhesion force of CNE-2Z cells was also increased accompanied with the increased E-cadherin expression. However, these results could not be observed in the NP69-SV40T cells even the concentration of DSF reached up to 400nM. Finally, the detection of cell wound scratch assay confirmed DSF-Cu could inhibit the migration of CNE-2Z cells, but no effect on NP69-SV40T cells. These findings demonstrated the selective cytotoxicity of DSF-Cu in CNE-2Z cells may attribute to the different mechanical properties and cytoskeleton rearrangement from the normal nasopharyngeal epithelial cells.

Motor neurons with limb-innervating character in the cervical spinal cord are sculpted by apoptosis based on the Hox code in chick embryo.

In the developing chick embryo, a certain population of motor neurons (MNs) in the non-limb-innervating cervical spinal cord undergoes apoptosis between embryonic days 4 and 5. However, the characteristics of these apoptotic MNs remain undefined. Here, by examining the spatiotemporal profiles of apoptosis and MN subtype marker expression in normal or apoptosis-inhibited chick embryos, we found that this apoptotic population is distinguishable by Foxp1 expression. When apoptosis was inhibited, the Foxp1+ MNs survived and showed characteristics of lateral motor column (LMC) neurons, which are of a limb-innervating subtype, suggesting that cervical Foxp1+ MNs are the rostral continuation of the LMC. Knockdown and misexpression of Foxp1 did not affect apoptosis progression, but revealed the role of Foxp1 in conferring LMC identity on the cervical MNs. Furthermore, ectopic expression of Hox genes that are normally expressed in the brachial region prevented apoptosis, and directed Foxp1+ MNs to LMC neurons at the cervical level. These results indicate that apoptosis in the cervical spinal cord plays a role in sculpting Foxp1+ MNs committed to LMC neurons, depending on the Hox expression pattern.

Berberine displays antitumor activity in esophageal cancer cells in vitro.

AIMTo investigate the effects of berberine on esophageal cancer (EC) cells and its molecular mechanisms.METHODSHuman esophageal squamous cell carcinoma cell line KYSE-70 and esophageal adenocarcinoma cell line SKGT4 were used. The effects of berberine on cell proliferation were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. For cell cycle progression, KYSE-70 cells were stained with propidium iodide (PI) staining buffer (10 mg/mL PI and 100 mg/mL RNase A) for 30 min and cell cycle was analyzed using a BD FACSCalibur flow cytometer. For apoptosis assay, cells were stained with an Annexin V-FITC/PI apoptosis detection kit. The rate of apoptotic cells was analyzed using a dual laser flow cytometer and estimated using BD ModFit software. Levels of proteins related to cell cycle and apoptosis were examined by western blotting.RESULTSBerberine treatment resulted in growth inhibition of KYSE-70 and SKGT4 cells in a dose-dependent and time-dependent manner. KYSE-70 cells were more susceptible to the inhibitory activities of berberine than SKGT4 cells were. In KYSE-70 cells treated with 50 μmol/L berberine for 48 h, the number of cells in G2/M phase (25.94% ± 5.01%) was significantly higher than that in the control group (9.77% ± 1.28%, P < 0.01), and berberine treatment resulted in p21 up-regulation in KYSE-70 cells. Flow cytometric analyses showed that berberine significantly augmented the KYSE-70 apoptotic population at 12 and 24 h post-treatment, when compared with control cells (0.83% vs 43.78% at 12 h, P < 0.05; 0.15% vs 81.86% at 24 h, P < 0.01), and berberine-induced apoptotic effect was stronger at 24 h compared with 12 h. Western blotting showed that berberine inhibited the phosphorylation of Akt, mammalian target of rapamycin and p70S6K, and enhanced AMP-activated protein kinase phosphorylation in a sustained manner.CONCLUSIONBerberine is an inhibitor of human EC cell growth and could be considered as a potential drug for the treatment of EC patients.

Thymoquinone induces apoptosis through downregulation of c-FLIP and Bcl-2 in renal carcinoma Caki cells.

Renal carcinoma is a common and frequently fatal carcinoma occurring worldwide and death rates due to this carcinoma are increasing with time. In the present study, we investigated the potential of thymoquinone a natural compound to induce apoptosis in renal carcinoma Caki cells. Thymoquinone efficiently enhanced the apoptotic population of Caki cells in a dose-dependent manner. Moreover, thymoquinone-mediated apoptosis caused downregulation of c-FLIP and Bcl-2, the master regulators of the anti-apoptotic mechanism. However, we did not find any changes in mRNA expression level of c-FLIP, therefore; this regulation of c-FLIP was a result of post-translation modification by thymoquinone. In contrast, expression of the Bcl-2 protein was observed at both transcriptional and translational level. However, we also observed that thymoquinone enhanced the level of intracellular reactive oxygen species (ROS) in Caki cells, which resulted in reduction of mitochondrial membrane potential (MMP) and cytochromec release into cytoplasm. Our results postulate that thymoquinone induces apoptosis through downregulating c-FLIP and Bcl-2 which can be utilized as a chemotherapeutic agent to treat renal carcinoma.

Lycopene inhibits the cell proliferation and invasion of human head and neck squamous cell carcinoma.

Lycopene has been shown to be associated with anticancer effects in numerous tumor types. However, the underlying mechanisms of lycopene in human head and neck squamous cell carcinoma (HNSCC) remain to be determined. The present study aimed to investigate the involvement of lycopene overload and the cytotoxic effects of lycopene on HNSCC cells, and to determine the possible mechanisms involved. Treatment with lycopene at a dose of >10 µM for >24 h inhibited the growth of FaDu and Cal27 cells in a time- and dose-dependent manner. The clearest increase in growth inhibition was due to the apoptotic population being significantly increased. The invasion abilities decreased with 25 µM lycopene exerting significant inhibitory effects (P<0.01). Mechanistic studies revealed that lycopene induced the upregulation of the pro-apoptotic protein, B-cell lymphoma-associated X protein, and therefore, resulted in the inhibition of the protein kinase B and mitogen-activated protein kinase signaling pathway. These data provided insights into the antitumor activity of lycopene in HNSCC cells.

By Changing Dimensionality, Sequential Culturing of Midbrain Cells, rather than Two-Dimensional Culture, Generates a Neuron-Glia Ratio Closer to in vivo Adult Midbrain

The neuron-glia ratio is of prime importance for maintaining the physiological homeostasis of neuronal and glial cells, and especially crucial for dopaminergic neurons because a reduction in glial density has been reported in postmortem reports of brains affected by Parkinson's disease. We thus aimed at developing an in vitro midbrain culture which would replicate a similar neuron-glia ratio to that in in vivo adult midbrain while containing a similar number of dopaminergic neurons. A sequential culture technique was adopted to achieve this. Neural progenitors (NPs) were generated by the hanging-drop method and propagated as 3D neurospheres followed by the derivation of outgrowth from these neurospheres on a chosen extracellular matrix. The highest proliferation was observed in neurospheres from day in vitro (DIV) 5 through MTT and FACS analysis of Ki67 expression. FACS analysis using annexin/propidium iodide showed an increase in the apoptotic population from DIV 8. DIV 5 neurospheres were therefore selected for deriving the differentiated outgrowth of midbrain on a poly-<smlcap>L</smlcap>-lysine-coated surface. Quantitative RT-PCR showed comparable gene expressions of the mature neuronal marker β-tubulin III, glial marker GFAP and dopaminergic marker tyrosine hydroxylase (TH) as compared to in vivo adult rat midbrain. The FACS analysis showed a similar neuron-glia ratio obtained by the sequential culture in comparison to adult rat midbrain. The yield of β-tubulin III and TH was distinctly higher in the sequential culture in comparison to 2D culture, which showed a higher yield of GFAP immunopositive cells. Functional characterization indicated that both the constitutive and inducible (KCl and ATP) release of dopamine was distinctly higher in the sequential culture than the 2D culture. Thus, the sequential culture technique succeeded in the initial enrichment of NPs in 3D neurospheres, which in turn resulted in an optimal attainment of the neuron-glia ratio on outgrowth culture from these neurospheres.

Naphthoflavones as Antiproliferative Agents: Design, Synthesis and Biological Evaluation.

The present study involves the design and synthesis of naphthoflavones as antiproliferative agents. The strategy presents naphthoflavones as hybrids of naphthyl based chalcones and flavones. A panel of human cancer cell lines were employed for the cytotoxicity studies. DK-13 exhibited significant cytoxicity against MiaPaCa-2 cell lines with IC50 value of 1.93 μM and 5.63 μM against MCF-7 cell lines. The compound DK-13 was found to induce apoptosis evidenced through phase contrast microscopy, DAPI staining, and mitochondrial membrane potential loss. The cell phase distribution studies indicated an increase from 11.26 % (control sample) to 55.19 % (sample treated with 20 μM compound DK-13) in the apoptotic population.

Influence of 6-Hydroxydopamine Toxicity on α-Synuclein Phosphorylation, Resting Vesicle Expression, and Vesicular Dopamine Release.

Post mortem studies on familial and sporadic Parkinson's disease patient striatal tissue have shown that nearly 90% of α-synuclein deposited in Lewy-bodies is phosphorylated at serine-129 (pSyn-129) as opposed to only 4% in normal human brain. We aimed to find the influence of endogenous neurotoxin 6-hydroxydopamine (6-OHDA) on α-synuclein phosphorylation, resting vesicles, and vesicular dopamine release. The relative distribution of pSyn-129+ cells in apoptotic and non-apoptotic populations at different 6-OHDA concentrations was assessed along with changes in oxidant-antioxidant system, mitochondrial membrane-potential, and intracellular-Ca2+ . Exposing SH-SY5Y cells to different concentrations of 6-OHDA for 48 h showed cell-death and apoptosis. Immunocytochemical analysis indicated an increase in pSyn-129 with increasing 6-OHDA concentration, and ELISA-estimation showed a significant increase in the pSyn-129 to α-synuclein ratio. FACS analysis also showed a significant increase in pSyn-129; and at sub-lethal 6-OHDA concentrations, pSyn-129+ cells were primarily distributed in the non-apoptotic population, suggesting that phosphorylation of α-synuclein precedes apoptosis. At higher 6-OHDA concentrations, the pSyn-129+ cell count significantly increased in the apoptotic population and decreased in the non-apoptotic population. Cytosolic co-localization of α-synuclein and ubiquitin was noticed at higher doses of 6-OHDA. FACS analysis showed decrease in vesicular monoamine transporter-2 (VMAT2) expression in 6-OHDA-treated cells, confirmed by reduction in functional dopamine-release on KCl and ATP stimulation. Significant decrease in VMAT2 expression and vesicular dopamine-release were observed with the lower 6-OHDA concentration, together with mild occurrence of apoptosis and significant increase in phosphorylated α-synuclein. This suggests that at sub-lethal 6-OHDA concentrations, the decrease in resting vesicles (VMAT2) and vesicular dopamine release are not attributable to apoptotic cell death and occur concomitantly with the phosphorylation of α-synuclein. J. Cell. Biochem. 117: 2719-2736, 2016. © 2016 Wiley Periodicals, Inc.

Quercetin induces caspase-dependent extrinsic apoptosis through inhibition of signal transducer and activator of transcription 3 signaling in HER2-overexpressing BT-474 breast cancer cells.

Flavonoids are assumed to exert beneficial effects in different types of cancers at high concentrations. Yet, their molecular mechanisms of action remain unknown. The present study aimed to examine the effect of quercetin on proliferation and apoptosis in HER2-expressing breast cancer cells. The anti-proliferative effects of quercetin were examined by proliferation, MTT and clonogenic survival assays. The effect of quercetin on expression of apoptotic molecules was determined by western blotting. Luciferase reporter assay was performed to measure signal transducer and activator of transcription 3 (STAT3) transcriptional activity. ELISA assay was performed to measure intracellular MMP-9 levels. Immunocytochemistry was performed to evaluate the nuclear STAT3 level. The results revealed that quercetin inhibited the proliferation of BT-474 cells in a dose- and time-dependent manner. Quercetin also inhibited clonogenic survival (anchorage-dependent and -independent) of BT-474 cells in a dose-dependent manner. These growth inhibitions were accompanied with an increase in sub-G0/G1 apoptotic populations. Quercetin induced caspase-dependent extrinsic apoptosis upregulating the levels of cleaved caspase-8 and cleaved caspase-3, and inducing the cleavage of poly(ADP-ribose) polymerase (PARP). In contrast, quercetin did not induce apoptosis via intrinsic mitochondrial apoptosis pathway since this compound did not decrease the mitochondrial membrane potential and did not affect the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (BAX). Quercetin reduced the expression of phospho-JAK1 and phospho-STAT3 and decreased STAT3-dependent luciferase reporter gene activity in the BT-474 cells. Quercetin inhibited MMP-9 secretion and decreased the nuclear translocation of STAT3. Our study indicates that quercetin induces apoptosis at concentrations >20 µM through inhibition of STAT3 signaling and could serve as a useful compound to prevent or treat HER2-overexpressing breast cancer.

Search for New Pro‐apoptotic agents in Indigenous Antitumor Plants in Nigeria

Due to severe cytotoxicities exacerbated by cytotoxic drugs, newer therapies from natural products are desirable. This work aimed to partially purify pro‐apoptotic agents from indigenous natural products and to investigate the mechanism of action of the selected plant fractions.Sixteen indigenous medicinal plants were collected and fractionated into hexane (HF), chloroform (CF), ethylacetate (EAF), methanol (MF) and water (WF) fractions using solvent‐solvent partitioning and polyamide adsorption chromatography. The resultant ninety‐six test fractions were screened for lethality potential using brine shrimp lethality (BSL) assay. Fractions with LC50 less than 10 ug/ml were subjected to Water Soluble Tetrazolium (WST‐1) cytotoxicity assay and apoptosis evaluations using: morphological assessment, flow cytometric analysis of phosphatidylserine externalization (Annexin V) and propidium iodide (PI) staining, caspase 3 enzyme assay, immunoblot assessment of Poly ADP‐Ribose Polymerase‐1 (PARP‐1) Cleavage, carboxymethyl‐2′,7′‐dichlorodihydrofluorescein‐diacetate (CM‐H2DCFDA) fluorescent microscopic/microplate analysis of reactive oxygen species generation, and Terminal deoxynucleotidyl transferase (Tdt) mediated dUTP‐biotin Nick End Labelling (TUNEL) assessment of DNA fragmentation.Following BSL assay, C. afer (EAF), P. guineense (HF), P. guineense (CF), A. virides (EAF), C. roseus (CF), Z. zanthoxyloides (HF) and Z. zanthoxyloides (EAF) had LC50 values &lt; 10 μg/ml. WST‐1 Cytotoxicity testing of these hit fractions on HeLa (Tumourigenic) and KMST‐6 (Non tumourigenic) cell Lines confirms their cytotoxic effects with the exception of Z. zanthoxyloides (HF). Significant (P&lt;0.05) selective cytotoxic preference for HeLa to KMST‐6 Cell lines was exhibited by P. guineense (CF) and C. roseus (CF). Membrane blebs were observed in HeLa cells treated with the hit fractions in comparison to 17.42 μg/ml(50μM) Camptothecin. Annexin V‐PI analysis reveals that Z. zanthoxyloides (EAF) and P. guineense (HF) showed similar apoptotic effect in comparison to 21.8% apoptotic population in Camptothecin. A significant (P&lt;0.05) increase in caspase‐3 enzyme activity was observed in C. roseus (CF) and Z. zanthoxyloides (EAF) in comparison with the negative control. PARP‐1 cleavage was confirmed in all the test groups except for A. virides (EAF) treated cells.Findings in this study suggest that all the hit test fractions elicited apoptotic effects but at varying degrees with Z. zanthoxyloides (EAF) showing the most prominent effect. While A. virides (EAF) elicits its cytotoxic effect via necrosis.

Caspase dependent apoptotic inhibition of melanoma and lung cancer cells by tropical Rubus extracts.

Rubus fairholmianus Gard. inhibits human melanoma (A375) and lung cancer (A549) cell growth by the caspase dependent apoptotic pathway. Herbal products have a long history of clinical use and acceptance. They are freely available natural compounds that can be safely used to prevent various ailments. The plants and plant derived products became the basis of traditional medicine system throughout the world for thousands of years. The effects of R. fairholmianus root acetone extract (RFRA) on the proliferation of A375 and A549 cells was examined in this study. RFRA led to a decrease in cell viability, proliferation and an increase in cytotoxicity in a dose dependent manner when compared with control and normal skin fibroblast cells (WS1). The morphology of treated cells supported apoptotic cell death. Annexin V/propidium iodide staining indicated that RFRA induced apoptosis in A375 and A549 cells and the percentages of early and late apoptotic populations significantly increased. Moreover, the apoptotic inducing ability of RFRA when analysing effector caspase 3/7 activity, indicated a marked increase in treated cells. In summary, we have shown the anticancer effects of RFRA in A375 and A549 cancer cells via induction of caspase dependent apoptosis in vitro. The extract is more effective against melanoma; which may suggest the usefulness of RFRA-based anticancer therapies.

Artemisinin inhibits proliferation of gallbladder cancer cell lines through triggering cell cycle arrest and apoptosis

To evaluate the effects of artemisinin on proliferation, cell cycle and apoptosis of gallbladder cancer cells. Gallbladder carcinoma cell lines(GBC-SD and NOZ)were cultured in vitro. The effects of artemisinin in different concentration on proliferation of the two cell lines in vitro were examined using MTT assay. The cell cycle distribution of GBC-SD and NOZ cells 24 h after treatments with artemisinin(20 μmol/L) were examined using flow cytometry. The apoptosis of GBC-SD and NOZ cells 24 h after treatments with artemisinin (20 μmol/L) were examined using Annexin V/PI staining.The expressions of p-ERK1/2, CDK4, cyclin D1, p16, cytochrome C and caspase-3 were examined by Western blot assay. t-test and one way ANOVA were used to evaluate the differences between two groups and more than two groups, respectively. The cell proliferation was significantly inhibited by artemisinin, the IC50 of artemisinin against GBC-SD and NOZ cells were 14.05 μmol/L and 12.42 μmol/L, respectively.Artemisinin induced cycle arrest, and G1 population of GBC-SD and NOZ cells increased to 74.60% and 78.86%. Cell apoptosis and apoptotic population of GBC-SD and NOZ cells were increased to 15.67% and 16.51% after dealt with artemisinin, respectively. In addition, expression of p16 was increased, and expressions of p-ERK1/2, CDK4 and cyclin D1 were down-regulated by artemisinin(all P<0.05). Cytochrome C was released from mitochondria to cytoplasm leading to the activation of caspase-3 and PARP after dealt with artemisinin(P<0.05). The inhibition effect of artemisinin on the proliferation gallbladder cancer cells is accompanied by down-regulation of ERK1/2 signaling pathway, G1 phase arrest and triggering caspase-3-mediate apoptosis.

Caspase dependent apoptotic activity of Rubus fairholmianus Gard. on MCF-7 human breast cancer cell lines

Developing plant derived chemopreventive agents that can divert the carcinogenic process and inhibit tumour progression may greatly reduce serious health consequences of cancer. This study investigated the antiproliferative and apoptosis inducing ability of Rubus fairholmianus root acetone (RFRA) on MCF-7 cells. RFRA treatments showed an increase in the in vivo antioxidant levels such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) in mice. The extract showed significant antitumour activity with 76.57% increase in life span of ascites tumour bearing mice and 1.94cm3 reductions in solid tumour volume at 250mg/kg. The cytotoxicity spectrum analysis indicated that RFRA is a potent cytocidal agent with varying levels of toxicity (IC50: 57.14–29.36μg/ml). A significant dose dependent decrease in viability, proliferation and increase in cytotoxicity, caspase 3/7 activity were observed in MCF-7 cells. RFRA (20μg/ml) induced apoptosis in MCF-7 cells, which was observed by morphology; DNA ladder formation and increased apoptotic population in flow cytometry analysis. These findings strongly suggest the use of R. fairholmianus as natural antioxidant with profound antitumour activities. It can act as a potent cytotoxic agent, which is able to induce apoptosis in MCF-7 cells via caspase 3/7 upregulation.

Physalin F, a seco-steroid from Physalis angulata L., has immunosuppressive activity in peripheral blood mononuclear cells from patients with HTLV1-associated myelopathy.

Human T-lymphotropic virus type 1 (HTLV-1) induces a strong activation of the immune system, especially in individuals with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Physalin F is a secosteroid with potent anti-inflammatory and immunomodulatory activities. The present study aimed to investigate the effects of physalin F on peripheral blood mononuclear cells (PBMC) of HAM/TSP subjects. A concentration-dependent inhibition of spontaneous proliferation of PBMC from HAM/TSP subjects was observed in the presence of physalin F, as evaluated by (3)H-thymidine uptake. The IC50 for physalin F was 0.97 ± 0.11 μM. Flow cytometry analysis using Cytometric Bead Array (CBA) showed that physalin F (10 μM) significantly reduced the levels of IL-2, IL-6, IL-10, TNF-α and IFN-γ, but not IL-17A, in supernatants of PBMC cultures. Next, apoptosis induction was addressed by using flow cytometry to evaluate annexin V expression. Treatment with physalin F (10 μM) increased the apoptotic population of PBMC in HAM/TSP subjects. Transmission electron microscopy analysis of PBMC showed that physalin F induced ultrastructural changes, such as pyknotic nuclei, damaged mitochondria, enhanced autophagic vacuole formation, and the presence of myelin-like figures. In conclusion, physalin F induces apoptosis of PBMC, decreasing the spontaneous proliferation and cytokine production caused by HTLV-1 infection.

Folate Receptor-Targeted Polymeric Micellar Nanocarriers for Delivery of Orlistat as a Repurposed Drug against Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a recalcitrant malignancy with no available targeted therapy. Off-target effects and poor bioavailability of the FDA-approved antiobesity drug orlistat hinder its clinical translation as a repurposed new drug against TNBC. Here, we demonstrate a newly engineered drug formulation for packaging orlistat tailored to TNBC treatment. We synthesized TNBC-specific folate receptor-targeted micellar nanoparticles (NP) carrying orlistat, which improved the solubility (70-80 μg/mL) of this water-insoluble drug. The targeted NPs also improved the delivery and bioavailability of orlistat to MDA-MB-231 cells in culture and to tumor xenografts in a nude mouse model. We prepared HEA-EHA copolymer micellar NPs by copolymerization of 2-hydroxyethylacrylate (HEA) and 2-ethylhexylacrylate (EHA), and functionalized them with folic acid and an imaging dye. Fluorescence-activated cell sorting (FACS) analysis of TNBC cells indicated a dose-dependent increase in apoptotic populations in cells treated with free orlistat, orlistat NPs, and folate-receptor-targeted Fol-HEA-EHA-orlistat NPs in which Fol-HEA-EHA-orlistat NPs showed significantly higher cytotoxicity than free orlistat. In vitro analysis data demonstrated significant apoptosis at nanomolar concentrations in cells activated through caspase-3 and PARP inhibition. In vivo analysis demonstrated significant antitumor effects in living mice after targeted treatment of tumors, and confirmed by fluorescence imaging. Moreover, folate receptor-targeted Fol-DyLight747-orlistat NP-treated mice exhibited significantly higher reduction in tumor volume compared to control group. Taken together, these results indicate that orlistat packaged in HEA-b-EHA micellar NPs is a highly promising new drug formulation for TNBC therapy. Mol Cancer Ther; 15(2); 221-31. ©2015 AACR.