Morphological Changes Of Apoptotic Cells Research Articles

P53 genotype-independent anticancer effects of olibanum extract and 11-keto-beta-boswellic acid on human colorectal cancer cells

BackgroundColorectal cancer (CRC) is the third most common cancer, and CRC-related mortality is increasing annually. A key regulator of the cell cycle and cell death, the p53 gene, is frequently found in patients with CRC as mutants. Since tumors with abnormal p53 sometimes show resistance to anticancer drugs, it is important to develop anticancer drugs according to genetic characteristics. Although evidence shows that boswellic acids (BAs) might be potential anticancer agents, the anticancer effects and mechanisms involved in their activities in CRC are unclear. Methods and ResultsHere, we investigated whether olibanum (Boswellia serrata Roxb.) extract and 11-keto-β-boswellic acid exerted cytotoxic effects against p53 wildtype, p53 mutated-, and p53-deleted human CRC cell lines. Results show that 11-keto-β-boswellic acid considerably reduced cell viability and induced cell cycle arrest in HCT116, HT29, and SW1417 cells. And increase of apoptotic cell population and morphological changes in apoptotic cells induced by 11-keto-β-boswellic acid were observed. The 11-keto-β-boswellic acid mediated cell cycle arrest and apoptosis were accompanied by changes in the expression of factors linked to apoptosis, regardless of p53 genotype. In addition, 11-keto-β-boswellic acid-treated cells dissociated from their spheroidal structure and appeared in an irregular form. ConclusionsAlthough further investigations are required to fully understand the underlying mechanisms, these results may provide insight into the potential of using olibanum extract and 11-keto-β-boswellic acid as therapeutic agents, with no difference in sensitivity depending on the p53 genotype.

Huaier inhibits autophagy and promotes apoptosis in T-cell acute lymphoblastic leukemia by down-regulating SIRT1

ObjectiveDue to the high drug resistance and relapse rate of T-cell acute lymphoblastic leukemia (T-ALL), the prognosis is usually poor. Therefore, there is an urgent need to find safer and more effective therapeutic drugs. Huaier and its preparations, as adjuvant drugs, have been widely used in the treatment of solid tumors and other diseases. However, the application of Huaier in leukemia is rarely reported. In this study, we investigated the anti-tumor effect of Huaier on T- ALL and its underlying mechanism. MethodsJurkat and MOLT-4 cells were treated with Huaier. Cell viability was evaluated by CCK-8 assay. The morphological changes of apoptotic cells were observed by Hoechst 33258 staining. Cell apoptosis was analyzed by flow cytometry. The expression levels of related proteins were assessed by Western blot. ResultsThe results showed that Huaier significantly inhibited the proliferation of Jurkat and MOLT-4 cells in a dose- and time-dependent manner, with IC50 of 2.37 ± 0.10 and 1.93 ± 0.07 mg/mL at 48 h, respectively. Morphological changes and increased number of apoptotic cells were observed by Hoechst 33258 staining and flow cytometry. The apoptosis rates of Jurkat and MOLT-4 cells in 4 mg/mL group were 50.67 ± 1.36 % and 49.97 ± 5.43 %, respectively. Huaier promoted the expression of Cytochrome c, Cleaved Caspase-3, Cleaved PARP, p53, LC3-Ⅱ and p62 proteins, while inhibited the expression of SIRT1, ATG7 and Beclin 1 proteins. Treatment with SRT1720 (SIRT1 agonist) combined with Huaier rescued Huaier-induced apoptosis and increased the expression of autophagy-related proteins. ConclusionHuaier inhibits autophagy and promotes apoptosis of T-ALL cells by down-regulating SIRT1, which may be a potential drug for the treatment of T-ALL.

Chemotherapeutic and Antiproliferative Effect of Purified Protein from Marine Catfish Tachysurus Dussumieri on Human Colon Cancer Cell Line.

The present study aimed to investigate the purified protein from the epidermal mucus of marine catfish Tachysurus dussumieri on the human colon cancer cell line. The bioactive protein was purified with the Anion exchange chromatography and the collected fractions were then tested to assess cell viability in HT 29 cells through the MTT assay. The most responding active purified protein fraction (PPF III) was characterized with the MALDI-TOF/MS it shared a similar homology and sequence with 90% of antimicrobial peptides from external secretions of amphibians. Typical morphological changes of apoptotic cells, including cell shrinkage and detachment, DNA damage, and nuclear condensation were observed after the treatment of bioactive protein. PPF III triggered ROS, increasing the LDH activity, disruption of mitochondrial membrane potential, and upregulation of Cleaved caspase 3/9, Cytochrome-c, Bax, and downregulation of Bcl-2 protein and gene expression on HT 29 cells.

Davidone C Induces the Death of Hepatocellular Carcinoma Cells by Promoting Apoptosis and Autophagy.

Davidone C is a newly discovered flavonoid compound purified from the ethyl acetate-soluble fraction of Sophora davidii (Franch.) Skeels. This study explored the anti-tumor activity of davidone C on hepatocellular carcinoma HepG2 and Bel-7402 cells and its mechanism through MTT method, morphological observation, flow cytometry and Western blotting. The results showed that davidone C significantly inhibited the proliferation of HepG2 and Bel-7402 cells in a time- and dose-dependent manner. The morphological changes of apoptotic cells can be observed under an inverted microscope, such as cell floating, chromosome condensation, apoptotic bodies, and other phenomena. The expressions of Bax, cleaved caspase-9, cleaved caspase-3 and cleaved PARP increased with the increase of dosage while Bcl-2 decreased, suggesting that the apoptotic mechanism might be related to the mitochondrial apoptotic pathway. Moreover, davidone C administration can down-regulate the expression of Grp78, and simultaneously up-regulate the expression of caspase-7 and caspase-12, indicating that the apoptotic mechanism might be related to the ERS pathway. In addition, davidone C can down-regulate the expression of p62, and simultaneously up-regulate the expression of LC3-I and LC3-II with a quantitative dependence, suggesting that the mechanism of apoptosis may be related to the autophagy signal pathway. All these results showed davidone C has potential effects on hepatocellular carcinoma.

Polymeric Lipid Hybrid Nanoparticles as a Delivery System Enhance the Antitumor Effect of Emodin in Vitro and in Vivo.

This study aimed to evaluate the therapeutic efficacy of Emodin-loaded polymer lipid hybrid nanoparticles (E-PLNs) for breast cancer. The size, Zeta potential, surface morphology, encapsulation efficiency, stability, in vitro drug release of E-PLNs prepared by the nanoprecipitation method were characterized. The uptake, in-vitro cytotoxicities and apoptosis of free drug, E-PLNs were investigated against MCF-7 cells. The efficacy of E-PLNs in tumor bearing nude mice has also been studied.The average particle size of the experimentally prepared E-PLNs was (122.7±1.79) nm, and the encapsulation rate was 72.8%. Compared with free Emodin (EMO), E-PLNs showed greater toxicity to MCF-7 cells by promoting the uptake of EMO, and can promote the early apoptosis of MCF-7 cells. In addition to the morphological changes of apoptotic cells, the ratio of Bax/Bcl-2 was significantly increased, which indicated that E-PLNs can induce apoptosis in MCF-7 cells to achieve anticancer effect. Finally, E-PLNs significantly inhibited tumor growth by more than 60%, which may be related to its passive targeting effect on tumor site. Our results suggest that E-PLNs have shown good anti-breast cancer effect than free EMO. Moreover, the effect of E-PLNs on MCF-7 cells is mainly related to the induction of apoptosis.

Rosiglitazone affects the progression of surgically‑induced endometriosis in a rat model.

Endometriosis is closely associated with inflammatory reactions and angiogenesis. Whether PPARγ is a target for the treatment of endometriosis remains unknown. The present study was designed to investigate the impact of a PPARγ agonist (rosiglitazone, RSG) on endometriosis in a rat model and to identify the underlying mechanism. The endometriosis model was established in rats. The pathological state of the endometrium was examined using hematoxylin-eosin staining. The microstructures of interest were visualized using electron microscopy. Western blot analysis and reverse transcription-quantitative polymerase chain reaction were used to detect PPARγ and MAT2A expression. VEGF and caspase-3 expression were investigated using immunohistochemistry. Pathological analysis revealed transparent and red nodules in the model group, and that vasoganglions were present all over the nodules. Endometrial epithelial hyperplasia was observed in the model group, and the shape was columnar. Increased interstitial cell numbers, with compact structure and abundant blood supply, were detected in the model group. Compared with the model group, incomplete epithelial structures with sparse interstitial cells and loose structure were observed in the pathological images from RSG treatment groups. Numerous inflammatory cells and poor blood supply were observed in the endometrial tissues, and the gland was filled mostly with vacuolar cells. Electron microscopy revealed that the tissue structure was integrated. Many vacuoles were formed within the endometrial tissue and the classical morphological changes of apoptotic cells were observed in RSG-treated groups. Caspase-3 and PPARγ expression increased and expression of VEGF and MAT2A decreased in RSG-treated groups. Taken together, these results revealed that RSG impacts the development and progression of endometriosis likely by inhibiting angiogenesis and inducing apoptosis.

Neuroprotective effects of urolithin A on H2O2-induced oxidative stress-mediated apoptosis in SK-N-MC cells.

BACKGROUND/OBJECTIVESOxidative stress causes cell damage and death, which contribute to the pathogenesis of neurodegenerative diseases. Urolithin A (UA), a gut microbial-derived metabolite of ellagitannins and ellagic acid, has high bioavailability and various health benefits such as antioxidant and anti-inflammatory effects. However, it is unknown whether it has protective effects against oxidative stress-induced cell death. We investigated whether UA ameliorates H2O2-induced neuronal cell death.MATERIALS/METHODSWe induced oxidative damage with 300 µM H2O2 after UA pretreatment at concentrations of 1.25, 2.5, and 5 µM in SK-N-MC cells. Cytotoxicity and cell viability were determined using the CCK-8 assay. The formation of reactive oxygen species (ROS) was measured using a 2,7-dichlorofluorescein diacetate assay. Hoechst 33342 staining was used to characterize morphological changes in apoptotic cells. The expressions of apoptosis proteins were measured using Western blotting.RESULTSUA significantly increased cell viability and decreased intracellular ROS production in a dose-dependent manner in SK-N-MC cells. It also decreased the Bax/Bcl-2 ratio and the expressions of cytochrome c, cleaved caspase-9, cleaved caspase-3, and cleaved PARP. In addition, it suppressed the phosphorylation of the p38 mitogen-activated protein kinase (MAPK) pathway.CONCLUSIONSUA attenuates oxidative stress-induced apoptosis via inhibiting the mitochondrial-related apoptosis pathway and modulating the p38 MAPK pathway, suggesting that it may be an effective neuroprotective agent.

Aspernolide A Inhibits the Proliferation of Human Laryngeal Carcinoma Cells through the Mitochondrial Apoptotic and STAT3 Signaling Pathways.

Aspernolide A, a butyrolactone secondary metabolite, was purified from the endophytic fungus Cladosporium cladosporioides derived from roots of Camptotheca acuminata Decne. In this study, the antitumor activity and mechanisms of aspernolide A on human laryngeal cancer Hep-2 and TU212 cells were studied by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, morphological observation and Western blotting. The results showed that aspernolide A significantly inhibited the proliferation of Hep-2 and TU212 cells in dose- and time-dependent manners. Morphological changes of apoptotic cells could be observed under an inverted microscope, such as irregular margins, decreased adherence ability and chromatin condensation. The expressions of Bax, Caspase-9, Caspase-3 and PARP (poly ADP-ribose polymerase) increased with the increase of dosage while Bcl-2 decreased, suggesting that the apoptotic mechanism might be related to the mitochondrial apoptotic pathway. Moreover, the expression of the phosphorylation of STAT3 decreased with the increase of dosage, suggesting that the apoptotic mechanism might be related to the STAT3 signaling pathway. All these conclusions indicated that aspernolide A has the potential anti-laryngocarcinoma effects.

Innovative Comprehensive Experiments to Study the Morphological Changes of Apoptotic Cells for Undergraduate Education.

This innovative comprehensive experiment is a compulsory course for third-year undergraduate students in the eight-year training program leading to a Doctor of Medicine degree or Doctor of Philosophy degree at Peking University Health Science Center (PKUHSC). There is a critical educational need to prepare these students for the increasing accessibility of research experience. This experimental curriculum of medical cell biology was developed to fulfill such a requirement, which consists of three experiments, including the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium) assay and the immunofluorescence and comet assays to observe antitumor drug-induced cell apoptosis. This experiment-based teaching system provides the participating students an opportunity to enhance their understanding of important biological research techniques and the instrumentation involved and to foster a better understanding of the research process within the classroom. Upon completion of the course, students' feedback showed that the curriculum increased their confidence in performing molecular techniques, and they reported positively on doing a research project in class. This course helped students improve their understanding of theoretical knowledge and actual research processes and contributed to their foundation for future research. © 2019 International Union of Biochemistry and Molecular Biology, 47(3):348-354, 2019.

Essential Oil from Carpesium abrotanoides L. Induces Apoptosis via Activating Mitochondrial Pathway in Hepatocellular Carcinoma Cells.

The effects of essential oil from Carpesium abrotanoides L. (CAEO) on the proliferation and apoptosis of human hepatic cancer cells were investigated in this study. MTT assays indicated that CAEO inhibited the proliferation of HCC cells with the IC50 values ranging from 41.28±3.06 to 130.36±20.79 μg/mL. Moreover, many obviously nuclear morphological changes of apoptotic cells in CAEO-treated HepG2 cells were detected by Hoechst 33258 staining and fluorescence microscopy. Flow cytometry was used to detect cell apoptosis and cell cycle, and noticeable findings showed that CAEO arrested cell-cycle at S and G2/M phases. The decreased Bcl-2/Bax protein ratio and the activation of caspase-3, caspase-9 were also detected by Western blotting. All results suggested that CAEO is a potential agent to fight against liver cancer, and the mitochondria-mediated intrinsic apoptotic pathway could be involved in CAEO-mediated apoptosis of human liver carcinoma cells.

Necrostatin-1 protects against ischemia/reperfusion injury by inhibiting receptor-interacting protein 1 in a rat flap model

The failure of reconstructive surgeries remains a challenge for plastic surgeons. Ischemia reperfusion (I/R) injury is considered to be one of the major problems in flap surgery. Necroptosis is a recently discovered and caspase-3-independent programed necrosis. Necrostatin-1 (Nec-1) is a specific inhibitor of necroptosis. Reports indicate that Nec-1 provides protection in ischemic models, such as brain, kidney, and heart. The aim of this study is to investigate the influence of Nec-1 on the I/R process in rat abdominal skin flaps. Twenty male Sprague-Dawley rats, weighing 280-320 g, were randomly divided into three groups. The extended epigastric skin flap (6 cm × 9 cm) of rats was used. Three hours of complete ischemia was performed using a clamp, and the clamp was then removed to reperfusion the flap. Twenty-four hours after the onset of the reperfusion, the rats were assessed for flap survival and perfusion analysis. One sample (1 cm × 1 cm) was taken for H&E, TUNEL, electron microscopy, IHC staining for RIP-1, and ELISA analysis for caspase-3 activity. Compared to the CTL group, the flap in the Nec-1 group showed a higher survival rate and better blood perfusion. In histological observation, skin flap in the Nec-1 group showed less inflammatory infiltration than the CTL group. The AI in the CTL group was higher than that in the Nec-1 group and showed typical morphological changes of apoptotic cells. In IHC study, RIP-1 expression was higher in the CTL group. But there was no significant difference between the two groups in caspase-3 activity detection. Nec-1 has a protective effect against I/R injury through the inhibition of RIP-1 on the skin flap model; this makes it a promising novel strategy in clinical setting.

Salidroside mitigates hypoxia/reoxygenation injury by alleviating endoplasmic reticulum stress‑induced apoptosis in H9c2 cardiomyocytes.

Endoplasmic reticulum (ER) stress-induced apoptosis serves a crucial role in the development of myocardial ischemia/reperfusion (I/R) injury. Salidroside is a phenylpropanoid glycoside isolated from Rhodiola rosea L., which is a plant often used in traditional Chinese medicine. It possesses multiple pharmacological actions and protects against myocardial I/R injury in vitro and in vivo. However, it is not yet clear whether ER stress or ER stress-induced apoptosis contributes to the cardioprotective effects of salidroside against myocardial I/R injury. Hence, hypoxia/reoxygenation (H/R)-treated H9c2 cardiomyocytes were used in the current study to mimic myocardium I/R injury in vivo. It was hypothesized that salidroside alleviates ER stress and ER stress-induced apoptosis, thereby reducing H/R injury in H9c2 cells. The results demonstrated that salidroside attenuated H/R-induced H9c2 cardiomyocyte injury, as cell viability was increased, lactate dehydrogenase release was decreased, morphological changes in apoptotic cells were ameliorated and the apoptosis ratio was reduced compared with the H/R group. ER stress was reversed, indicated by the downregulation of glucose regulated protein 78 and C/EBP homologous protein following pretreatment with salidroside. In addition, salidroside attenuated ER stress-induced apoptosis, as the expression of cleaved caspase-12 and pro-apoptotic protein Bcl-2 associated X protein and activity of caspase-3 was decreased, while the expression of anti-apoptotic protein Bcl-2 was increased following pretreatment with salidroside. Furthermore, the results indicated that salidroside decreases the activation of the ER stress-associated signaling pathway, as the expression of phosphorylated protein kinase RNA (PKR)-like ER kinase (p-PERK) and phosphorylated inositol-requiring enzyme-1α (p-IRE1α) proteins were decreased following pretreatment with salidroside. These results demonstrate that salidroside protects against H/R injury via regulation of the PERK and IRE1α pathways, resulting in alleviation of ER stress or ER stress-induced apoptosis in H9c2 cardiomyocytes.

Anticancer effects of a novel chroman analog in HeLa cells are associated with G2‑phase arrest and mitochondrial‑mediated apoptosis.

In the present study, the anticancer activity of 1‑[(3S,4R)‑2,2‑dimethyl‑3‑oxo‑4‑(2‑piperidonyl)chroman‑6‑yl]‑3‑phenylurea (S32) was investigated by testing its effect in vitro on the growth of HeLa cells. First, we showed that the IC50 value of S32 was ~70 µM by using WST‑8 assay, and that it significantly inhibited the proliferation and viability of HeLa cells in a dose‑dependent manner after 48h. Morphological changes in apoptotic cells included cellular shrinkage and nuclear condensation. The results of [3H]‑thymidine incorporation and flow cytometric analysis indicated that S32 induced inhibition of DNA replication and G2‑phase cell cycle arrest. Moreover, S32 induced the levels of reactive oxygen species (ROS) and decreased the mitochondrial membrane potential (MMP) in a time‑dependent manner. Using Annexin V‑FITC/propidium iodide (PI) dual staining assay, we found that S32 noticeably increased early apoptosis in HeLa cells in a time‑dependent manner. The result of western blot analysis showed that the apoptotic induction was associated with an increase in Bax levels and a decrease in Bcl‑2 levels, which led to activation of caspase‑8, ‑9and‑3. Taken together, our findings demonstrated that S32 induces mitochondrial‑mediated apoptosis in HeLa cells and suggest that S32 has potential as an anticancer drug.

Chinese herbal medicine Euphorbia esula extract induces apoptosis and inhibits the proliferation, migration and invasion of multidrug resistant gastric carcinoma cells

This paper aims to study the effects of traditional Chinese medicine Euphorbia esula on multidrug resistant human gastric cancer cells in the cell proliferation, migration, invasion and apoptosis, and to study the apoptosis-inducing pathway. Different dilutions of Euphorbia esula extract were used to process human multidrug resistant gastric cancer SGC7901/ADR cells. Cell proliferation inhibition phenomenon was determined by MTT experiment. Nuclear morphological changes of apoptotic cells and apoptotic indexes were observed and determined by Hochest33528 staining followed with fluorescence microscope observing. Flow cytometry was used to detect cell apoptosis rate. Cell migration and invasion ability were observed and determined by Transwell method. Spectrophotometry was used to detect caspase-3 and caspase-9 enzyme activity. Western blotting was used to detect subcellular distribution of cytochrome c. The results showed that Euphorbia esula extract had obvious inhibition effect on proliferation of gastric cancer multidrug resistant SGC7901/ADR cells, which was time- and concentration-dependent. After processing multidrug resistant gastric cancer SGC7901/ADR cells with Euphorbia esula extract, the apoptotic index and apoptosis rate were significantly increased than those in the control group, which showed a time- and dose-dependent mode; but if a caspase inhibitor was added, apoptosis index was not obviously increased. Transwell method showed that migration and invasion ability of the Euphorbia esula extract-processed SGC7901/ADR cells dropped significantly. Spectrophotometry showed that in Euphorbia esula extract-processed SGC7901/ADR cells, caspase-3 and caspase-9 expression were increased, which had significant differences with the control group. Western blotting test showed that the distribution of cytochrome c decreased in mitochondria, while increased in the cytoplasm (i.e., cytochrome c escaped from mitochondria to the cytoplasm). In conclusion, Euphorbia esula extract could inhibit the proliferation, migration and invasion, and induce apoptosis in human gastric cancer multidrug resistant SGC7901/ADR cells; and cytochrome c, caspase-9 and caspase-3 might be involved in cell apoptosis induced by Euphorbia esula extract, suggesting endogenous or mitochondrial apoptotic pathway.

Quantitative analysis and comparison of 3D morphology between viable and apoptotic MCF-7 breast cancer cells and characterization of nuclear fragmentation.

Morphological changes in apoptotic cells provide essential markers for defining and detection of apoptosis as a fundamental mechanism of cell death. Among these changes, the nuclear fragmentation and condensation have been regarded as the important markers but quantitative characterization of these changes is yet to be achieved. We have acquired confocal image stacks of 206 viable and apoptotic MCF-7 cells stained by three fluorescent dyes. Three-dimensional (3D) parameters were extracted to quantify and compare their differences in morphology. To analyze nuclear fragmentation, a new method has been developed to determine clustering of nuclear voxels in the reconstructed cells due to fluorescence intensity changes in nuclei of apoptotic cells. The results of these studies reveal that the 3D morphological changes in cytoplasm and nuclear membranes in apoptotic cells provide sensitive targets for label-free detection and staging of apoptosis. Furthermore, the clustering analysis and morphological data on nuclear fragmentation are highly useful for derivation of optical cell models and simulation of diffraction images to investigate light scattering by early apoptotic cells, which can lead to future development of label-free and rapid methods of apoptosis assay based on cell morphology.

Tectorigenin attenuates the MPP+-induced SH-SY5Y cell damage, indicating a potential beneficial role in Parkinson's disease by oxidative stress inhibition.

Tectorigenin is a plant isoflavonoid primarily derived from the flowers of Pueraria thomsonii Benth. Although various biological properties of tectorigenin have been reported, such as its antioxidant activity, the effects of tectorigenin on the cellular models of Parkinson's disease have not yet been elucidated. The aims of the current study were to investigate whether tectorigenin prevents neurotoxicity induced by MPP+ (also known as 1-methyl-4-phenylpyridinium) in SH-SY5Y cells and to elucidate the underlying protective mechanism. Cell viability and lactate dehydrogenase release were measured. The morphological changes of apoptotic cells were observed by Hoechst 33258 staining. Caspase-3, superoxide dismutase, catalase and glutathione peroxidase activity was measured using commercially available ELISA kits. The expression of cytochrome c, Bax, Bcl-2 and NADPH oxidase were detected by western blot analysis. The results indicated that treatment with MPP+ causes a significant decrease in the viability of cells and an increase in apoptosis, as evidenced by the upregulation of apoptotic cells, caspase-3 activity and cytochrome c expression. By contrast, these effects were all reversed by pretreatment with tectorigenin in SH-SY5Y cells. Tectorigenin also inhibited the MPP+-induced changes of Bax and Bcl-2 levels. In addition, pretreatment with tectorigenin mitigated the MPP+-caused increases in the levels of reactive oxygen species and NADPH oxidase protein in SH-SY5Y cells. Simultaneously, tectorigenin abolished the downregulation of antioxidant enzymes, including superoxide dismutase, catalase and glutathione peroxidase, that was induced by MPP+. In conclusion, the present study data indicate that the neuroprotective effect of tectorigenin against MPP+-induced cytotoxicity and apoptosis may be involved in attenuating oxidative stress and enhancing antioxidant defense.

Fangchinoline inhibits migration and causes apoptosis of human breast cancer MDA-MB-231 cells.

In order to improve outcomes after breast cancer treatment, it is essential to understand the mechanisms of action of potential therapeutic agents. The effect of fangchinoline (FAN) on migration and apoptosis of human breast cancer MDA-MB-231 cells and its underlying mechanisms were investigated. MDA-MB-231 cells were treated with different concentrations of FAN, growth inhibition rates were measured by MTT assay and morphological changes of apoptotic cells were observed by Hoechst staining. The wound-healing assay was used to determine of the effect of FAN on the migration of MDA-MB-231 cells. ELISA was used to detect the expression of MMP-2 and −9 in MDA-MB-231 cells treated with different concentrations of FAN and western blot analysis was used to quantify expression of NF-κβ and Iκβ proteins in the same cells. Our results showed that FAN significantly inhibited the growth of MDA-MB-231 cells in concentration-dependent manner and it induced MDA-MB-231 cell apoptosis. With the high FAN concentrations and long exposure times, the levels of MMP-2 and −9 decreased and the expression of NF-κβ decreased, while the expression of Iκβ protein increased. Based on these results, the antitumor effects of FAN on breast cancer cells can be explained at least partially by inducing apoptosis and inhibiting the migration of MDA-MB-231 cells.

The Chinese herb Prunella vulgaris promotes apoptosis in human well-differentiated thyroid carcinoma cells via the B-cell lymphoma-2/Bcl-2-associated X protein/caspase-3 signaling pathway.

Prunella vulgaris (PV), a traditional Chinese herb, has been shown to be rich in bioactive chemicals and possess anti-proliferative and pro-apoptotic effects on tumor cells. The effect of PV on human well-differentiated thyroid carcinoma (WDTC), which accounts for the majority of common endocrine malignancies, remains to be elucidated. The present study aimed to investigate the function of PV on WDTC cell lines and apoptosis-associated signaling pathway activity. Additional studies demonstrated that PV may induce apoptosis in WDTC TPC-1 and FTC-133 cell lines, using the Cell Counting Kit-8 assay. Morphological changes of apoptotic cells were observed by Hoechst 33342 and acridine orange/ethidium bromide staining. In addition, ladder pattern of fragmented DNA was observed by DNA gel electrophoresis. It was also observed that PV significantly increased Bcl-2-associated X protein and caspase-3 expression, and downregulated B-cell lymphoma-2 expression in TPC-1 and FTC-133 by reverse transcription-quantitative polymerase chain reaction (P<0.05). Thus, the present results indicated that PV has the potential to be a future WDTC therapeutic agent.

Essential oil from Siegesbeckia pubescens induces apoptosis through the mitochondrial pathway in human HepG2 cells

Siegesbeckia pubescens (SP) has been used as a traditional medicine for the treatment of and inflammatory diseases. However, the activities of SP against hepatocellular carcinoma and the related mechanisms remain unclear. The present study aimed to examine the effects of the essential oil of SP (SPEO) on the proliferation of hepatocellular carcinoma cells and the possible mechanisms. The growth inhibition of HepG2 cells was analyzed by MTT assay. Hoechst 33258 and fluorescence microscopy were utilized to observe the nuclear morphological changes of apoptotic cells. Flow cytometry was used to detect cell apoptosis and cell cycle. The expressions of the target proteins were detected by Western blotting. The results showed that SPEO obviously inhibited the proliferation of HepG2 cells in a dose-dependent manner. SPEO activated a series of apoptotic proteins in HepG2 cells, increasing expression levels of Bax, caspase-3 and caspase-9, and decreasing the bcl-2 expression level. SPEO displayed promising anti-hepatocellular carcinoma activities in vitro, partly by inducing apoptosis in HepG2 cells through activating the mitochondrial pathway.

Synergistic Effect of Forbesione From Garcinia hanburyi in Combination with 5-Fluorouracil on Cholangiocarcinoma

Background: Chemotherapy for advanced cholangiocarcinoma (CCA) is largely ineffective; thus innovative combinations of chemotherapeutic agents and natural compounds represent a promising strategy. This study aimed to investigate the synergistic effects of forbesione combined with 5-fluorouracil (5-FU) in hamster cholangiocarcinoma (Ham-1) cells both in vitro and in vivo. The anti-tumor effects of 5-FU combined with forbesione in vitro were determined using the Sulforhodamine B (SRB) assay and the effects in vivowere assessed in transplanted Ham-1 allograph models. Using ethidium bromide/acridine orange (EB/AO) staining, the morphological changes of apoptotic cells was investigated. The expressions of apoptosis-related molecules after combined treatment with forbesione and 5-FU were determined using real-time RT-PCR and western blot analysis. Forbesione or 5-FU alone inhibited proliferation of Ham-1 cells in a dose-dependent manner and their combination showed a synergistic proliferation inhibitory effect in vitro. In vivostudies, forbesione in combination with 5-FU exhibited greater inhibition of the tumor in the hamster model compared with treatment using either drug alone. Forbesione combined with 5-FU exerted stronger apoptotic induction in Ham-1 cells than did single drug treatment. The combination of drugs strongly suppressed the expression of B-cell lymphoma 2 (Bcl-2) and procaspase-3 while enhancing the expression of p53, Bcl-2-associated X protein (Bax), apoptotic protease activating factor-1 (Apaf-1), caspase-9 and caspase-3, compared with single drug treatments. These results explained the decreased expression of cytokeratin 19 (CK19) positive cells and proliferation cell nuclear antigen (PCNA) positive cells in Ham-1 cell tumor tissues of the treated hamsters. There was no apparent systemic toxicity observed in the treated animals compared with the control groups. Forbesione combined with 5-FU strongly induced apoptosis in Ham-1 cells. The growth inhibitory effect of combined treatment using these two drugs was much greater than treatment with either drug alone, both in vitroand in vivo.