Effects of 5,6-Dihydroxy-2,4-Dimethoxy-9,10-Dihydrophenanthrene on G2/M Cell Cycle Arrest and Apoptosis in Human Lung Carcinoma Cells.

5,6-dihydroxy-2,4-dimethoxy-9,10-dihydrophenanthrene (HMP) is an active compound isolated from the rhizome extracts of Dioscorea membranacea Pierre, a Thai medicinal plant. This study aimed to investigate the growth-inhibitory and apoptosis-inducing effects of HMP in human lung cancer A549 cells. The antiproliferative and cytotoxic effects of HMP were analyzed by a Sulforhodamine B assay. Cell division, cell cycle distribution and membrane asymmetry changes were each performed with different fluorescent dyes and then analyzed by flow cytometry. Real-time PCR and immunoblotting were used to detect cell cycle- and apoptosis-related mRNA levels and proteins, respectively. The nuclear morphology of the cells stained with DAPI and DNA fragmentation were detected by fluorescence microscopy and gel electrophoresis, respectively. The results showed that HMP exerted strong antiproliferative and cytotoxic activities in A549 cells with the highest selectivity index. It halted the cell cycle in [Formula: see text]/M phase via down-regulation of the expression levels of regulatory proteins Cdc25C, Cdk1 and cyclinB1. In addition, HMP induced early apoptotic cells with externalized phosphatidylserine and subsequent apoptotic cells in sub-[Formula: see text] phase. HMP increased caspase-3 activity and levels of the cleaved (active) form of caspase-3 whose actions were supported by the cleavage of its target PARP, nuclear condensation and DNA apoptotic ladder. Moreover, HMP significantly increased the mRNA and protein levels of proapoptotic Bax as well as promoted subsequent caspase-9 activation and BID cleavage, indicating HMP-induced apoptosis via both intrinsic and extrinsic pathways. These data support, for the first time, the potential role of HMP as a cell-cycle arrest and apoptosis-inducing agent for lung cancer treatment.

mRNA Expression of Bax, Bcl-2, p53, Cathepsin B, Caspase-3 and Caspase-9 in the HepG2 Cell Line Following Induction by a Novel Monoclonal Ab Hep88 mAb: Cross-Talk for Paraptosis and Apoptosis.

Monoclonal antibodies with specific antigens have been widely used as targeted therapy for cancer. Hep88 mAb is a monoclonal antibody which shows specific binding with anti-cancer effects against the HepG2 cell line. However, its mechanisms of action are still not completely understood. We examined cell cycling and apoptosis by flow cytometry and mRNA expression of factors involved in apoptosis and paraptosis in Hep88 mAb-treated HepG2 cells by real-time PCR. The cell-cycle analysis demonstrated that growth-inhibitory activity was associated with G2/M cell cycle arrest. Hep88 mAb induced a significant increase in apoptotic cell populations in a dose- and time-dependent manner. The mRNA expression results also suggested that the process triggered by Hep88 mAb involved up-regulation of tumor suppressor p53, pro-apoptotic Bax, Cathepsin B, Caspase-3 and Caspase-9, with a decrease of anti-apoptotic Bcl-2 - thus confirming paraptosis and apoptosis programmed cell death. These findings represent new insights into the molecular mechanisms underlying the anti-cancer properties of Hep88 mAb in liver cancer cells.

Hep88 mAb-mediated paraptosis-like apoptosis in HepG2 cells via downstream upregulation and activation of caspase-3, caspase-8 and caspase-9.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death worldwide. Presently, targeted therapy via monoclonal antibodies to specific tumor-associated antigens is being continuously developed. Hep88 mAb has proven to exert tumoricidal effects on the HepG2 cell via a paraptosis-like morphology. To verify the pathway, we then demonstrated downstream up-regulation of caspase-3, caspase-8 and caspase-9, assessingmRNA expression by real-time PCR and associated enzyme activity by colorimetric assay. Active caspase-3 determination was also accomplished by flow cytometry. Active caspase-3 expression was increased by Hep88 mAb treatment in a dose-and time-dependent manner. All of the results indicated that Hep88 mAb induced programmed cell death in the HepG2 cell line from paraptosis-like to apoptosis by downstream induction of caspases. These conclusions imply that Hep88mAb might be a promising tool for the effective treatment of HCC in the future.

Growth arrest and apoptosis via caspase activation of dioscoreanone in human non-small-cell lung cancer A549 cells.

BACKGROUND: Dioscoreanone (DN) isolated from Dioscorea membranacea Pierre has been reported to exert potent cytotoxic effects against particular types of cancer. The present study was carried out to investigate the cytotoxicity of DN against a panel of different human lung cancer cell lines. The study further examined the underlying mechanisms of its anticancer activity in the human lung adenocarcinoma cell line A549. METHODS: Antiproliferative effects of DN were determined by SRB and CFSE assays. The effect of DN on cell cycle distribution was assessed by flow cytometric analysis. Apoptotic effects of DN were determined by sub-G1 quantitation and Annexin V-FITC/PI flow cytometric analyses, as well as by changes in caspase-3 activity and relative levels of Bax and Bcl-2 mRNA. RESULTS: DN exerted antiproliferative and cytotoxic effects on all three subtypes of non-small cell lung cancer (NSCLC) cells, but not on small cell lung cancer (SCLC) cells and normal lung fibroblasts. DN slowed down the cell division and arrested the cell cycle at the G2/M phase in treated A549 cells, leading to a dose- and time- dependent increase of the sub-G1 population (apoptotic cells). Consistently, early apoptotic cells (AnnexinV +/PI-) were detected in those cells that were treated for 24 h and increased progressively over time. Moreover, the highest activity of caspase-3 in DN-treated A549 cells was detected within the first 24 h, and pretreatment with the general caspase inhibitor z-VAD-fmk completely abolished such activity and also DN-induced apoptosis in a dose-dependent manner. Additionally, DN increased the Bax/Bcl-2 ratio in treated A549 cells with time, indicating its induction of apoptosis via the mitochondrial pathway. CONCLUSIONS: This study reveals for the first time that the anticancer activity of DN was induced through regulation of the Bcl-2 family protein-mediated mitochondrial pathway and the subsequent caspase-3 activation in A549 cancer cells, thus supporting its potential role as a natural apoptosis-inducing agent for NSCLC.

Crocodylus siamensis serum and macrophage phagocytic activity.

Antimicrobial activity of sera from many crocodilian species has been recognized. This activity was proposed to be mediated, at least in part, by complement. Due to the fact that complement proteins have different functions in the immune system, they may be involved in phagocytic process of phagocytes. In the present study, the effects of Siamese crocodile serum on phagocytic activity of macrophages as well as the possible involvement of complement in this process were examined. The results showed increases in the phagocytosis of both Escherichia coli and to a lesser extent, Staphylococcus aureus upon incubation of murine macrophage cell line with fresh crocodile serum (FS). Similar to FS, other crocodile blood products, including freeze dried serum (DS) and freeze dried whole blood (DWB) exhibited phagocytosis-enhancing property. However the ability of DWB to enhance phagocytosis was less efficient than that of FS and DS, suggesting that serum factors were involved in this process. Treatment of FS with heat at 56 degrees C for 30 min deteriorated the effect of FS on bacterial uptake of macrophages, suggesting that complement proteins play a role in the modulation of the phagocytic process. Collectively, the results of the present study suggested that crocodile serum enhances the macrophage phagocytic activity through complement activity and, therefore, may be taken as an alternative medicine for supporting the human immune responses.

Prevalence and risk factors for pinworm infection in the kindergarten of Thammasat University, Thailand.

We studied the prevalence and risk factors for pinworm infection in children attending the kindergarten of Thammasat University, Pathum Thani, Thailand, using the Scotch-tape technique. Slides were examined by a standard light microscope; 20% of negative slides were reexamined for quality control. Symptoms and risk factor data were collected using a structured questionnaire. Three hundred thirty children age 3 to 6 years old were sampled (males=159). Sixty-five (19.7%) had symptoms consistent with pinworm infection. No pinworm eggs were detected. Most parents (73%) had a good socioeconomic status and 64% were university graduates. Pinworm infection may be uncommon in urban Thailand.