Antitumor activity of ginsenoside Rd in gastric cancer via up-regulation of Caspase-3 and Caspase-9.

Ginsenoside Rd (GS-Rd), isolated from the Chinese traditional herbal medicine Panax ginseng, is used for the treatment of cardiovascular diseases, inflammation, different body pains, and trauma. Caspase-3 and Caspase-9 belong to cysteine aspartic acid specific protease (Caspase) family that plays an important role in apoptosis progression of cancers. In the present study, we investigated the anti-tumor effect of GS-Rd by MTT assay, colony formation assessment, flow cytometry, and Western blotting. Our results revealed that ginsenoside Rd significantly inhibits human gastric cancer (GC) growth and cell proliferation. Flow cytometer analysis showed that the GS-Rd could significantly induce apoptosis and arrest the G0/G1 phase in GC cells. Further, GS-Rd was found to increase the ratio of Bax/Bcl-2 and the expression of Caspase-3 and Caspase-9, respectively, and to decrease the expression of Cyclin D1. Taken together, our study suggests that GS-Rd significantly inhibits GC cell proliferation, induces cell apoptosis through increase the expression of Caspase-3, Caspase-9, and the ratio of Bax/Bcl-2. GS-Rd also induces cell cycle arrest at G0/G1 phase by down-regulation Cyclin D1. Thus, GS-Rd could serve as a lead to develop novel therapeutic agents to against human gastric cancer.

Amiloride Alleviates Neurological Deficits Following Transient Global Ischemia and Engagement of Central IL-6 and TNF-α Signal.

BACKGROUND: Central pro-inflammatory cytokine (PIC) signal is involved in neurological deficits after transient global ischemia induced by cardiac arrest (CA). The present study was to examine if blocking acid sensing ion channels (ASICs) using amiloride in the Central Nervous System can alleviate neurological deficits after the induction of CA and further examine the participation of PIC signal in the hippocampus for the effects of amiloride. METHODS: CA was induced by asphyxia and then cardiopulmonary resuscitation was performed in rats. Western blot analysis and ELISA were used to determine the protein expression of ASIC subunit ASIC1 in the hippocampus, and the levels of PICs. As noted, it is unlikely that this procedure is clinically used although amiloride and other pharmacological agents were given into the brain in this study. RESULTS: CA increased ASIC1 in the hippocampus of rats in comparison with control animals. This was associated with the increase in IL-1ß, IL-6 and TNF-α together with Caspase-3 and Caspase-9. The administration of amiloride into the lateral ventricle attenuated the upregulation of Caspase-3/Caspase-9 and this further alleviated neurological severity score and brain edema. Inhibition of central IL-6 and TNF-α also decreased ASIC1 in the hippocampus of CA rats. CONCLUSION: Transient global ischemia induced by CA amplifies ASIC1a in the hippocampus likely via PIC signal. Amiloride administered into the Central Nervous System plays a neuroprotective role in the process of global ischemia. Thus, targeting ASICs (i.e., ASIC1a) is suggested for the treatment and improvement of CA-evoked global cerebral ischemia.

Induction of Apoptosis in HepaRG Cell Line by Aloe-Emodin through Generation of Reactive Oxygen Species and the Mitochondrial Pathway.

BACKGROUND/AIMS: Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-anthraquinone), an anthraquinone active compounds, is isolated from some traditional medicinal plants such as Rheum palmatum L. and Cassia occidentalis, which induce hepatotoxicity in rats. The aim of this study was to determine potential cytotoxic effects of aloe-emodin on HepaRG cells and to define the underlying mechanism. METHODS: MTT was used to evaluate cell viability. Apoptotic cell death was analyzed via Annexin V-FITC/PI double staining. Intracellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were determined by flow cytometry, while the expression of apoptosis-related proteins was determined by Western blot analysis. RESULTS: Treatment with aloe-emodin significantly reduced cell viability and induced apoptosis in HepaRG cells in a dose- and time-dependent manner. It provoked ROS generation and depolarization of MMP in HepaRG cells when compared with controls. Aloe-emodin dose-dependently increased release of mitochondrial cytochrome c, and levels of Fas, p53, p21, Bax/Bcl-2 ratio, as well as activation of caspase-3, caspase-8, caspase-9, and subsequent cleavage of poly(ADP-ribose)polymerase (PARP). It also induced S-phase cell cycle arrest by increasing the expression of p21 and cyclin E proteins while significantly decreasing the expression of cyclin A and CDK2. CONCLUSION: These results suggest that aloe-emodin inhibits cell proliferation and induces apoptosis in HepaRG cells, most probably through a mechanism involving both Fas death pathway and the mitochondrial pathway by generation of ROS. These findings underscore the need for risk assessment of human exposure to aloe-emodin.

Phytol induces ROS mediated apoptosis by induction of caspase 9 and 3 through activation of TRAIL, FAS and TNF receptors and inhibits tumor progression factor Glucose 6 phosphate dehydrogenase in lung carcinoma cell line (A549).

A number of drugs as well as lead molecules are isolated from natural sources. Phytol is one of such lead molecule belongs to terpenes group distributed widely in medicinal plants. In the present work, we investigated the cytotoxic behavior of phytol on human lung carcinoma cells (A549). Phytol was found to cause characteristic apoptotic morphological changes and generation of ROS in A549 cells. The mechanism of phytol involved the activation of TRAIL, FAS and TNF-α receptors along with caspase 9 and 3. In silico molecular docking studies revealed that phytol has a good binding affinity with glucose-6-phosphate dehydrogenase (G6PD), which is known to promote tumor proliferation. The ability of phytol to become potential drug candidate has been revealed from the pharmacokinetic study performed in the present study.

Chemosensitizing Effect of Astragalus Polysaccharides on Nasopharyngeal Carcinoma Cells by Inducing Apoptosis and Modulating Expression of Bax/Bcl-2 Ratio and Caspases.

BACKGROUND Platinum-based chemotherapy is the most effective regimen for nasopharyngeal carcinoma, which presents highly invasive and metastatic activity. However, the dose-related toxicity of chemotherapy agents limits the dose administration. Astragalus polysaccharide (APS) is the major active ingredient extracted from Chinese herb Radix Astragali and is proven to be active against carcinomas. We aimed to assess the chemosensitizing effects of Astragalus polysaccharides on nasopharyngeal carcinoma in vitro and in vivo and to explore the underlying mechanism. MATERIAL AND METHODS We used BALB/c nu/nu mice and human nasopharyngeal carcinoma cell lines CNE-1, CNE-2, and SUNE-1. MTT, Annexin V/PI, Western blot analysis, and TUNEL assay were carried out. RESULTS APS significantly promoted anti-proliferative and apoptotic effects of cisplatin on nasopharyngeal carcinoma cells. APS also enhanced the anti-tumor effects and cisplatin-induced apoptosis in the xenograft model. The level of Bcl-2 decreased, while the levels of Bax, caspase-3, and caspase-9 increased in cisplatin combined with APS treatment compared to cisplatin only treatment. The ratio of Bax to Bcl-2 was significantly enhanced by the APS to cisplatin. CONCLUSIONS APS enhanced the anti-proliferative and apoptotic effect of cisplatin by modulating expression of Bax/Bcl-2 ratio and caspases on nasopharyngeal carcinoma cells and in the xenograft model.

YiXin-Shu, a ShengMai-San-based traditional Chinese medicine formula, attenuates myocardial ischemia/reperfusion injury by suppressing mitochondrial mediated apoptosis and upregulating liver-X-receptor α.

Positive evidence from clinical trials has fueled growing acceptance of traditional Chinese medicine (TCM) for the treatment of cardiac diseases; however, little is known about the underlying mechanisms. Here, we investigated the nature and underlying mechanisms of the effects of YiXin-Shu (YXS), an antioxidant-enriched TCM formula, on myocardial ischemia/reperfusion (MI/R) injury. YXS pretreatment significantly reduced infarct size and improved viable myocardium metabolism and cardiac function in hypercholesterolemic mice. Mechanistically, YXS attenuated myocardial apoptosis by inhibiting the mitochondrial mediated apoptosis pathway (as reflected by inhibition of mitochondrial swelling, cytochrome c release and caspase-9 activity, and normalization of Bcl-2 and Bax levels) without altering the death receptor and endoplasmic reticulum-stress death pathways. Moreover, YXS reduced oxidative/nitrative stress (as reflected by decreased superoxide and nitrotyrosine content and normalized pro- and anti-oxidant enzyme levels). Interestingly, YXS upregulated endogenous nuclear receptors including LXRα, PPARα, PPARß and ERα, and in-vivo knockdown of cardiac-specific LXRα significantly blunted the cardio-protective effects of YXS. Collectively, these data show that YXS is effective in mitigating MI/R injury by suppressing mitochondrial mediated apoptosis and oxidative stress and by upregulating LXRα, thereby providing a rationale for future clinical trials and clinical applications.

Recombinant Human Keratinocyte Growth Factor Induces Akt Mediated Cell Survival Progression in Emphysematous Mice.

INTRODUCTION: Emphysema has been associated with decreased VEGF and VEGFR-2 expression and the presence of high numbers of apoptotic alveolar cells. Keratinocyte growth factor stimulates VEGF synthesis which in turn confers normal lung structure maintenance via the Akt pathway. In this study the potential role of rHuKGF in the improvement of deregulated Akt mediated cell survival pathway in emphysematous mice was investigated. METHODS: Three experimental groups, i.e., emphysema, treatment and control groups, were prepared. Lungs of mice were treated on 3 occasions by oropharyngeal instillation of 10mg rHuKGF per kg body weight after induction of emphysema with porcine pancreatic elastase. Subsequently, lung tissues from mice were collected for histopathology and molecular biology studies. RESULTS AND DISCUSSION: Histopathology photomicrographs and destructive index analysis have shown that elastase-induced airspace enlargement and loss of alveoli recovered in the treatment group. rHuKGF stimulates VEGF production which in turn induces the Akt mediated cell survival pathway in emphysematous lungs. mRNA expression of VEGF, VEGFR, PI3K and Akt was significantly increased while Pten, Caspase-9 and Bad was notably decreased in treatment group when compared with emphysema group, being comparable with the control group. Moreover, VEGF protein expression was in accordance with that found for mRNA. CONCLUSION: Therapeutic rHuKGF supplementation improves the deregulated Akt pathway in emphysema, resulting in alveolar cell survival through activation of the endogenous VEGF-dependent cell survival pathway. Hence rHuKGF may prove to be a potential drug in the treatment of emphysema.

Roles of p53 and caspases in induction of apoptosis in MCF- 7 breast cancer cells treated with a methanolic extract of Nigella sativa seeds.

BACKGROUND: Nigella Sativa (NS) is an herb from the Ranunculaceae family that exhibits numerous medicinal properties and has been used as important constituent of many complementary and alternative medicines (CAMs). The ability of NS to kill cancer cells such as PC3, HeLa and hepatoma cells is well established. However, our understanding of the mode of death caused by NS remains nebulous. The objective of this study was to gain further insight into the mode and mechanism of death caused by NS in breast cancer MCF-7 cells. MATERIALS AND METHODS: Human breast cancer cells (MCF-7) were treated with a methanolic extract of NS, and a dose- and time-dependent study was performed. The IC50 was calculated using a Cell Titer Blue® viability assay assay, and evidence for DNA fragmentation was obtained by fluorescence microscopy TUNEL assay. Gene expression was also profiled for a number of apoptosis-related genes (Caspase-3, -8, -9 and p53 genes) through qPCR. RESULTS: The IC50 of MCF-7 cells was 62.8 µL/mL. When MCF-7 cells were exposed to 50 µL/mL and 100 µL/mL NS for 24 h, 48 h and 72 h, microscopic examination (TUNEL assay) revealed a dose- and time-dependent increase in apoptosis. Similarly, the expression of the Caspase-3, -8, -9 and p53 genes increased significantly according to the dose and time. CONCLUSIONS: NS induced apoptosis in MCF-7 cells through both the p53 and caspase pathways. NS could potentially represent an alternative source of medicine for breast cancer therapy.

Isoquercitrin inhibits the progression of liver cancer in vivo and in vitro via the MAPK signalling pathway.

Liver cancer is a malignant tumour with high morbidity and fatality rates that is common worldwide. At present, the clinical approaches to treating primary liver cancer include partial hepatectomy, systemic or local chemotherapy, radiotherapy, radiofrequency ablative surgery and liver transplantation. However, all of these approaches have shortcomings, including poor prognosis and numerous side-effects. A large number of studies have proven that many effective ingredients in traditional Chinese medicine, particularly the flavonoid compounds extracted from plants, have achieved breakthroughs in terms of enhancing the effects and reducing the toxicity of chemotherapy and radiotherapy, preventing tumour metastasis and relapse after surgery, alleviating the clinical symptoms of advanced tumours, improving the quality of life of the patient with tumours and extending patient long­term survival. The purpose of the present study was to investigate the impact of isoquercitrin, the flavonoid from Bidens bipinnata L. extract, on the progression of liver cancer and to achieve a deeper understanding of the biological characteristics of isoquercitrin's involvement in the progression of liver cancer. In the in vitro experiments, isoquercitrin was found to strongly inhibit the proliferation of human liver cancer cells, promote the apoptosis of human liver cancer cells, and block the cell cycle in the G1 phase. Isoquercitrin activated caspase-3, -8 and -9, inhibited the expression level of ERK and p38MAPK protein phosphorylation, and promoted the phosphorylation of JNK. Additionally, isoquercitrin reduced the expression level of PKC in human liver cancer cells. In the in vivo experiments, isoquercitrin was also found to significantly inhibit the growth of transplanted tumours in nude mice. The present study confirmed that isoquercitrin could inhibit the progression of human liver cancer in vivo and in vitro, and the molecular mechanism of isoquercitrin may be closely associated with the MAPK and PKC signalling pathways.

Triptolide induces apoptotic cell death of multiple myeloma cells via transcriptional repression of Mcl-1.

Triptolide, a diterpenoid trioxide purified from the Chinese herb Tripterygium wilfordii Hook F, has been used as a natural medicine in China for hundreds of years. Several reports have demonstrated that triptolide inhibits the proliferation of cancer cells in vitro and reduces the growth of several types of tumors in vivo. To address the potential of triptolide as a novel therapeutic agent for patients with multiple myeloma, we investigated the effects of triptolide on the induction of apoptosis in human multiple myeloma cells in vitro. Triptolide rapidly induces apoptotic cell death in various myeloma cell lines. Triptolide-induced apoptosis in myeloma cells is associated with the loss of mitochondrial transmembrane potential (∆ψm), the release of cytochrome c and Smac/DIABLO from mitochondria into the cytosol, and the activation of caspase-3 and caspase-9. Furthermore, triptolide induces a rapid decline in the levels of Mcl-1 protein that correlates with caspase activation and induction of apoptosis. Inhibition of Mcl-1 synthesis by triptolide occurs at the level of mRNA transcription and is associated with an inhibition of phosphorylation of RNA polymerase II CTD. These results indicate that Mcl-1 is an important target for triptolide-induced apoptosis in myeloma cells that occurs via inhibition of Mcl-1 mRNA transcription coupled with rapid protein degradation through the ubiquitin-proteasome pathway.

Alteration of Bax/Bcl-2 ratio contributes to Terminalia belerica-induced apoptosis in human lung and breast carcinoma.

The objective of the present study was to assess the in vitro anticancer activity of 70% methanolic extract of Terminalia belerica (TBME) against human lung (A549) and human breast (MCF-7) carcinoma and its possible mechanism. TBME showed significant cytotoxicity to both A549 and MCF-7 cells, whereas, no cytotoxicity was found in non-malignant WI-38 cells. Flow cytometric analysis was then performed and 100 µg/ml of TBME was selected as the effective concentration inducing apoptosis in A549 and MCF-7. At this concentration, TBME caused DNA fragmentation pattern of apoptosis. Furthermore, mechanism of apoptosis induction was demonstrated using western blotting and Bax/Bcl-2 ratio in both types of the cells was found increased, which leads to the activation of caspase cascade along with the cleavage of PARP. These results suggested that TBME is able to induce anticancer effects on both lung and breast cancer cell lines through the modulation of Bcl-2 family proteins.

Induction of G2/M phase cell cycle arrest and apoptosis by ginsenoside Rf in human osteosarcoma MG­63 cells through the mitochondrial pathway.

Ginsenosides, extracted from the traditional Chinese herb ginseng, are a series of novel natural anticancer products known for their favorable safety and efficacy profiles. The present study aimed to investigate the cytotoxicity of ginsenoside Rf to human osteosarcoma cells and to explore the anticancer molecular mechanisms of ginsenoside Rf. Five human osteosarcoma cell lines (MG-63, OS732, U-2OS, HOS and SAOS-2) were employed to investigate the cytotoxicity of ginsenoside Rf by MTT and colony forming assays. After treatment with ginsenoside Rf, MG-63 cells which were the most sensitive to ginsenoside Rf, were subjected to flow cytometry to detect cell cycle distribution and apoptosis, and nuclear morphological changes were visualized by Hoechst 33258 staining. Caspase-3, -8 and -9 activities were also evaluated. The expression of cell cycle markers including cyclin B1 and Cdk1 was detected by RT-PCR and western blotting. The expression of apoptotic genes Bcl-2 and Bax and the release of cytochrome c were also examined by western blotting. Change in the mitochondrial membrane potential was observed by JC-1 staining in situ. Our results demonstrated that the cytotoxicity of ginsenoside Rf to these human osteosarcoma cell lines was dose-dependent, and the MG-63 cells were the most sensitive to exposure to ginsenoside Rf. Additionally, ginsenoside Rf induced G2/M phase cell cycle arrest and apoptosis in MG-63 cells. Furthermore, we observed upregulation of Bax and downregulation of Bcl-2, Cdk1 and cyclin B1, the activation of caspase-3 and -9 and the release of cytochrome c in MG-63 cells following treatment with ginsenoside Rf. Our findings demonstrated that ginsenoside Rf induces G2/M phase cell cycle arrest and apoptosis in human osteosarcoma MG-63 cells through the mitochondrial pathway, suggesting that ginsenoside Rf, as an effective natural product, may have a therapeutic effect on human osteosarcoma.

Effect of α, ß momorcharin on viability, caspase activity, cytochrome c release and on cytosolic calcium levels in different cancer cell lines.

A multitude of plants have been used extensively for the treatment of cancers throughout the world. The protein, α, ß momorcharin has been extracted from the plant Momordica charantia (MC), and it possesses anti-cancer and anti-HIV properties similar to the crude water and methanol soluble extract of the plant. This study investigated the anti-cancer effects and the cellular mechanisms of action of α, ß momocharin (200-800 µM) on 1321N1, Gos-3, U87-MG, Sk Mel, Corl-23 and Weri Rb-1 cancer cell lines compared to normal healthy L6 muscle cell line measuring cell viability using MTT assay kit, Caspase-3 and 9 activities, cytochrome c release and intracellular free calcium concentrations [Ca(2+)]i. The results show that α, ß momorcharin can evoke significant dose-dependent (P < 0.05; Student's t test) decreases in the viability (increases in cell death) of 1321N1, Gos-3, U87-MG, Sk Mel, Corl-23 and Weri Rb-1 cancer cell lines compared to healthy L6 muscle cell line and untreated glioma cells. α, ß momorcharin (800 µM) also evoked significant (P < 0.05) increases in caspase-3 and 9 activities and cytochrome c release. Similarly, α, ß momorcharin elicited significant (P < 0.05) time-dependent elevation in [Ca(2+)]i in all five glioma cell lines compared to untreated cells. Together, the results have demonstrated that α, ß momorcharin can exert its anti-cancer effect on different cancer cell lines by intracellular processes involving an insult to the mitochondria resulting in cellular calcium over loading, apoptosis, cytochrome release and subsequently, cell death.

Induction of caspase-9, biochemical assessment and morphological changes caused by apoptosis in cancer cells treated with goniothalamin extracted from Goniothalamus macrophyllus.

Goniothalamin, a natural compound extracted from Goniothalamus sp. belonging to the Annonacae family, possesses anticancer properties towards several tumor cell lines. This study focused on apoptosis induction by goniothalamin (GTN) in the Hela cervical cancer cell line. Cell growth inhibition was measured by MTT assay and the IC50 value of goniothalamin was 3.2 ± 0.72 µg/ml. Morphological changes and biochemical processes associated with apoptosis were evident on phase contrast microscopy and fluorescence microscopy. DNA fragmentation, DNA damage, caspase-9 activation and a large increase in the sub-G1 and S cell cycle phases confirmed the occurrence of apoptosis in a time-dependent manner. It could be concluded that goniothalamin show a promising cytotoxicity effect against cervical cancer cells (Hela) and the cell death mode induced by goniothalamin was apoptosis.

Apoptosis of K562 leukemia cells by Abnobaviscum F®, a European mistletoe extract.

Evidence suggests that mistletoe extract has the potential to be used as an anticancer agent. Abnobaviscum F® is a European mistletoe extract from the host tree Fraxinus. We investigated the effect of Abnobaviscum F on the growth and survival of different leukemia cell lines. Abnobaviscum F treatment strongly reduced survival and induced apoptosis of K562 (human myeloid leukemia), RPMI-8226 (human plasmacytoma) and L1210 (murine lymphocytic leukemia) cells in culture. Using K562 cells to further investigate the mechanism of action of Abnobaviscum F, we showed that Abnobaviscum F-induced cell death was associated with the activation of caspase-9, JNK-1/2 and p38 MAPK, as well as with the downregulation of Mcl-1, and inhibition of ERK-1/2 and PKB phosphorylation. Moreover, Abnobaviscum F treatment led to both a reduction of cellular glutathione (GSH) and the induction of ER stress (GRP78 and CHOP induction and eIF-2α phosphorylation). By contrast, Abnobaviscum F did not impact the expression of the DR4 and DR5 death receptors. The Abnobaviscum F-induced apoptosis of K562 cells was blocked by pretreatment with either GSH, z-VAD-fmk or SP600125. Our results, therefore, show that Abnobaviscum F induces apoptosis of K562 cells through the activation of the intrinsic caspase pathway, the phosphorylation of JNK-1, the reduction of cellular GSH, and the induction of ER stress.

Methanol extract of the ethnopharmaceutical remedy Smilax spinosa exhibits anti-neoplastic activity.

Plants have been the source of several effective drugs for the treatment of cancer and over 60% of anticancer drugs originate from natural sources. Therefore, extracts of the rhizome of Smilax spinosa, an ethnomedicinal plant from Guatemala which is used for the treatment of inflammatory conditions, were investigated regarding their anti-neoplastic activities. By using several solvents the methanol extract was by far the most potent against HL60 cell proliferation (50% inhibition at 60 µg/ml). Furthermore, fractionation of this extract yielded fraction F2, which exhibited enforced pro-apoptotic activity, and activated CYP1A1. Proteins that are relevant for cell cycle progression and apoptosis, as well as proto-oncogenes were investigated by western blotting. This revealed that the methanol extract increased the levels of p21 and this may have caused cell cycle attenuation. The derivative fraction F2 induced apoptosis through the intrinsic pathway, which correlated with the inhibition of Stat3 phosphorylation and concomitant induction of caspase 9, then caspase 8 and caspase 3. In summary, the methanol extract and the derivative fraction F2 of S. spinosa showed anti-neoplastic effects in HL-60 cells and CYP1A1 activation in estrogen receptor-positive MCF-7 breast cancer cells but not in estrogen-negative MDA-MB231 breast cancer cells. Based on our data Smilax spinosa may be a promising source for novel anticancer agents.

Preferential inhibition of hepatocellular carcinoma by the flavonoid Baicalein through blocking MEK-ERK signaling.

Baicalein is a purified flavonoid extracted from the roots of Scutellaria baicalensis or Scutellaria radix. Although previous studies have suggested that Baicalein possesses an in vitro anti-hepatocellular carcinoma activity, its in vivo effects and mechanisms of action are still not completely understood. In this study, Baicalein at concentrations of 40-120 µM exhibited significant cytotoxicity to three hepatocellular carcinoma (HCC) cell lines but marginal cytotoxicity to a normal liver cell line in vitro. Compared to a standard chemotherapy drug, 5-fluorouracil (5-FU), Baicalein had greater effect on HCC cells but less toxicity on normal liver cells. Treatment with Baicalein dramatically reduced mitochondrial transmembrane potential, and activated caspase-9 and caspase-3. Blockade of Baicalein-induced apoptosis with a pan-caspase inhibitor partially attenuated Baicalein-induced growth inhibition in HCC. Baicalein treatment significantly inhibited tumor growth of HCC xenografts in mice. Induction of apoptosis was demonstrated in Baicalein-treated xenograft tumors by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. Furthermore, Baicalein treatment dramatically decreased the levels of phosphorylation of MEK1, ERK1/2 and Bad in vitro and in vivo. Overexpression of human MEK1 partially blocked Baicalein-induced growth inhibition. Consequently, these findings suggest that Baicalein preferentially inhibits HCC tumor growth through inhibition of MEK-ERK signaling and by inducing intrinsic apoptosis.

Norcantharidin triggers cell death and DNA damage through S-phase arrest and ROS-modulated apoptotic pathways in TSGH 8301 human urinary bladder carcinoma cells.

Norcantharidin (NCTD) is one of the ingredients of blister beetles which have been used in Chinese medicine for a long time. The purpose of this study was to investigate the inhibitory effects of NCTD on TSGH 8301 human bladder cancer cells in vitro and the mechanisms through which it exerts its anticancer action. Cell morphological analysis was performed using a phase-contrast microscope. The percentage of viable cells, cell cycle distribution, sub-G1 phase (apoptosis), reactive oxygen species (ROS) production and the levels of mitochondrial membrane potential (∆Ψ(m)) were analyzed by flow cytometry. DNA condensation and damage were determined by DAPI staining and comet assay. Apoptosis-associated protein level changes in TSGH 8301 cells following exposure to NCTD were examined, measured and determined by western blotting. Analysis of protein translocation was conducted by immunostaining and confocal laser microscopy. The results indicated that NCTD promoted cytotoxic effects, including the induction of cell morphological changes and the decrease in the percentage of viability, the induction of S-phase arrest as well as sub-G1 phase (apoptosis) in TSGH 8301 cells. The activities of caspase-3 and -9 were upregulated following NCTD treatment. Western blotting indicated that NCTD upregulated Fas, FasL, Bax, Bid, cytochrome c, caspase-3, -8 and -9 that led to the induction of apoptosis through the Fas extrinsic pathway. Furthermore, NCTD induced AIF and Endo G that were released from mitochondria to induce apoptosis through the mitochondrial-independent pathway. NCTD upregulated ROS production, downregulated ∆Ψ(m) and ERK, JNK, p38 protein kinases in TSGH 8301 cells. These findings suggest that NCTD triggers apoptosis in TSGH 8301 human bladder cancer cells via the Fas receptor, activation of the caspse-8, -9 and -3, mitochondrial-dependent and -independent pathways. NCTD may be useful for developing new therapeutic regimens for the treatment of bladder cancer.

Involvement of the mitochondrial pathway in bruceine D-induced apoptosis in Capan-2 human pancreatic adenocarcinoma cells.

The fruit of Brucea javanica L. is a common herb used in Chinese medicine for the treatment of a variety of cancers. Our research group has previously identified bruceine D (BD), a quassinoid found abundantly in B. javanica, to have potent cytotoxic effect on a number of pancreatic cancer cell lines, including Panc-1, SW1990 and Capan-1 cells. In the present study, we showed that BD was also able to inhibit the growth of the Capan-2 human pancreatic adenocarcinoma cell line, but it exerted only modest cytotoxicity on the WRL68 human hepatocyte cell line and a human pancreatic progenitor cell line. The antiproliferative effects of BD were comparable to those exhibited by camptothecin and gemcitabine in our culture system. We found a dose-dependent decrease of the mitochondrial membrane potential in BD-treated Capan-2 cells as measured by the JC-1 assay. BD exposure was able to attenuate the expression of Bcl-2 protein in Capan-2 cells as detected by western blot analysis. In addition, the expression of both caspase 9 and caspase 3 in BD-treated Capan-2 cells was significantly accentuated. Moreover, BD was capable of inducing the fragmentation of genomic DNA in Capan-2 cells as evidenced by Hoechst staining. Cell cycle analysis demonstrated that BD could increase the percentage of Capan-2 cells in the subG1 phase in a dose-related manner. An increase in the apoptosis of Capan-2 cells was also observed by Annexin V and PI staining. These results unequivocally indicate that BD induces cytotoxicity in Capan-2 cells via the induction of cellular apoptosis involving the mitochondrial pathway.