benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and tanshinone

Tanshinone IIA (T2A), a derivative of phenanthrenequinone and also the major active ingredient of Danshen, has been paid extensive attention as a promising cancer therapy for its potential anti-cancer activities. In this study, the apoptosis and autophagy of human prostate cancer PC-3 cells were observed after 5 μM T2A treatment, as well as their relevance. Mitochondrial-dependent apoptosis was firstly detected through morphological observation and biochemical analysis. Meanwhile, 5 μM T2A successfully triggered the autophagy of PC-3 cells, indicated by increased expression of Beclin1, and LC3 II. Validation experiments were conducted to further consolidate T2A's contribution to autophagy: Pretreatment with autophagy inhibitor 3-methyladenine (3-MA) provided protection against autophagy and enhanced T2A-induced apoptosis. Besides, the apoptosis suppressor z-VAD-fmk failed to facilitate the formation of autophagic vacuoles, which also proved the T2A-induced autophagy independent of apoptosis. Moreover, the reactive oxygen species (ROS) scavenger N-acetyl-L-cysteine (NAC) efficiently inhibited the expression of Beclin1, LC3-II, and cleaved caspase-3, which indicated apoptosis and autophagy with dependence on intracellular ROS production. Taken together, these results demonstrated that autophagy is the cytoprotective mechanism in this experimental system, and the ROS resulted from T2A treatment played a critical role in apoptosis and autophagy initiation.

Topics: Abietanes; Acetylcysteine; Adenocarcinoma; Amino Acid Chloromethyl Ketones; Antineoplastic Agents, Phytogenic; Apoptosis; Autophagy; Beclin-1; Caspase 3; Cell Line, Tumor; Drug Screening Assays, Antitumor; Humans; Male; Microtubule-Associated Proteins; Neoplasm Proteins; Prostatic Neoplasms; Reactive Oxygen Species

Natural compounds isolated from medicinal plants are invaluable resources for drug discovery. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent unique by its cancer cell-specific proapoptotic action, but its potential is heavily curbed by acquired resistance. We herein reported for the first time the identification of cytotoxic and TRAIL-sensitizing components of Salvia miltiorrhiza (Danshen), a traditional medicinal plant effective for treating cardiovascular disorders. Specifically, we found that the ethanol extract and its group 5 fraction of S. miltiorrhiza showed evident cytotoxicity against the human lung adenocarcinoma cell line A549 and ovarian adenocarcinoma cell line TOV-21G in a concentration-dependent manner. Likewise, a dose-dependent cytotoxicity was exerted by the standard solutions of cryptotanshinone, tanshinone I and tanshinone IIA, the major components of the group 5 fraction, where tanshinone IIA were most potent and displayed an IC₅₀ of 2.00 ± 0.36 μM and 2.75 ± 0.23 μM for A549 and TOV-21G, respectively. Induction of apoptosis represents an essential mechanism underlying tanshinone IIA-mediated cytotoxic action, as evidenced by the proteolytic processing of PARP upon tanshinone IIA stimulation and, importantly, a marked rescue of the viability of tanshinone IIA-treated cells when co-treatment with the pan-caspase inhibitor z-VAD-fmk. Noteworthy, stimulation with cryptotanshinone, tanshinone I or tanshinone IIA all effectively potentiated TRAIL to reduce viability and inhibit the colony formation capacity of TRAIL-resistant TOV-21G and SKOV3. Collectively, we revealed the proapoptotic and TRAIL-sensitizing components of S. miltiorrhiza and further implicated the potential of developing these active compounds as monotherapeutic agent or TRAIL-based therapy for cancer chemoprevention or chemotherapy.

Topics: Abietanes; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Cell Division; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Female; Humans; Inhibitory Concentration 50; Neoplasms; Phenanthrenes; Phenanthrolines; Plant Extracts; Poly(ADP-ribose) Polymerases; Salvia miltiorrhiza; TNF-Related Apoptosis-Inducing Ligand

Tanshinone IIA (Tan-IIA) is extracted from Danshen, Salviae miltiorrhizae Radix, which has been widely adopted in traditional herbal medicine to treat cardiovascular and hepatic diseases. Tan-IIA induces apoptosis and inhibits proliferation in human hepatocellular carcinoma (HCC) cells. However, the molecular mechanisms of Tan-IIA on human HCC cells are not understood clearly. In the present study, the cytotoxicity of Tan-IIA as well as its molecular mechanisms in human HCC J5 cells was investigated. The cytotoxicity was assayed by MTT. The protein expression of p53, p21, Bax, Bcl-2, Cdc25c, Cdc2, calreticulin, caspase 12, GADD153, caspase 3 and beta-actin in J5 cells were determined by Western blotting. The cell cycles were analyzed by FACS. The protein expression of caspase 12, GADD1533 and caspase 3 were detected by immunocytochemical staining. The results showed that Tan-IIA inhibited J5 cells in a dose- and time-dependent manner. The protein expression of p53, p21, Bax, calreticulin, caspase 12, caspase 3 and GADD153 were increased, but Bcl-2, Cdc25c and Cdc2 were decreased in J5 cells. In addition, the results also showed that Tan-IIA arrested J5 cells in the G2/M phase. Immunocytochemistry staining showed that J5 cells treated with Tan-IIA up-regulated the protein expression of caspase 12, 3 and GADD153. Taken together, the findings suggest that Tan-IIA inhibits and induces apoptosis in J5 cells through novel molecular targets, calreticulin, caspase 12 and GADD153.

Topics: Abietanes; Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Calreticulin; Carcinoma, Hepatocellular; Caspase 12; Caspase Inhibitors; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cysteine Proteinase Inhibitors; Drugs, Chinese Herbal; Humans; Liver Neoplasms; Male; Mitochondria; Phenanthrenes; Transcription Factor CHOP

Tanshinone IIA, a major component extracted from the traditional herbal medicine, Salvia miltiorrhiza Bunge, is known to exhibit potent cytotoxicity against various human carcinoma cells in vitro. However, the mechanism by which tanshinone IIA produces this anti-tumor effect remains unknown. Since anti-neovascularization has generally been regarded as an effective strategy for anti-cancer therapy, we decided to investigate the mechanism underlying tanshinone IIA-mediated death of human endothelial cells. In this study, we demonstrate that tanshinone IIA elicits human endothelial cell death independent of oxidative stress. These events are partially calcium-dependent and actually dependent upon NAD(P)H: quinone oxidoreductase (NQO1) activity. Tanshinone IIA induces an increase in intracellular calcium, which triggers the release of cytochrome c, thus causing loss of the mitochondrial membrane potential (MMP), resulting in the subsequent activation of caspases. Blocking the induction of Ca2+ perturbation with BAPTA-AM partially rescued cells from tanshinone IIA-induced cytotoxicity. Additionally, blocking NQO1 activity with dicoumoral or inhibiting caspase activities with the general caspase inhibitor, z-VAD-fmk, prevented cell death induced by tanshinone IIA. Therefore, our results imply that tanshinone IIA-mediated cytotoxicity against human endothelial cells may occur through activation of NQO1, which induces a calcium imbalance and mitochondrial dysfunction, thus stimulating caspase activity.

Topics: Abietanes; Acridine Orange; Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; Blotting, Western; Calcium; Caspase Inhibitors; Caspases; Cell Cycle; Cell Death; Cytochromes c; Dicumarol; Drugs, Chinese Herbal; Egtazic Acid; Electrophoresis, Polyacrylamide Gel; Endothelial Cells; Enzyme Activation; Enzyme Inhibitors; Humans; Membrane Potentials; Mitochondria; Models, Biological; NAD(P)H Dehydrogenase (Quinone); Oxidative Stress; Phenanthrenes; Plant Extracts; Reactive Oxygen Species; Salvia miltiorrhiza; Time Factors