In Vitro Cytotoxic Activity of Clinacanthus nutans Leaf Extracts Against HeLa Cells

Objective: This study was conducted to investigate the antiproliferative activity of extracts of Clinacanthus nutansleaves against human cervical cancer (HeLa) cells. Methods: C. nutans leaves were subjected to extraction using 80%methanol or water. The methanol extract was further extracted to obtain hexane, dichloromethane (DCM), and aqueousfractions. The antiproliferative activity of the extracts against HeLa cells was determined. The most cytotoxic extractwas furthered analyzed by apoptosis and cell cycle assays, and the phytochemical constituents were screened by gaschromatography-mass spectrometry (GC-MS). Results: All of the extracts were antiproliferative against HeLa cells, andthe DCM fraction had the lowest IC50 value of 70 μg/mL at 48 h. Microscopic studies showed that HeLa cells exposedto the DCM fraction exhibited marked morphological features of apoptosis. The flow cytometry study also confirmedthat the DCM fraction induced apoptosis in HeLa cells, with cell cycle arrest at the S phase. GC-MS analysis revealedthe presence of at least 28 compounds in the DCM fraction, most of which were fatty acids. Conclusion: The DCMfraction obtained using the extraction method described herein had a lower IC50 value than those reported in previousstudies that characterized the anticancer activity of C. nutans against HeLa cells.     

Bioactivity Guided Fractionation and Elucidation of Anti-Cancer Properties of Imperata Cylindrica Leaf Extracts

Background: In our earlier study, we reported the anticancer effect of methanolic extracts of, I. cylindrica leaf (ICL) against human oral squamous cell carcinoma cell lines SCC-9. The cytotoxic effect of ICL methanolic extract was specific to the cancer cells and not to the normal cells. The present study aimed to fractionate the ICL methanolic extract to derive anticancer bioactives. Methods: The ICL methanolic extract was subjected to a bioactivity guided fractionation. Cytotoxic, cell cycle inhibitory, apoptosis and caspase gene expression inducing activity of the active fractions were evaluated using MTT assay, FACS analysis, Annexin V binding assay and RT-PCR respectively. Results: The hexane fraction of ICL methanolic extract (ICLH) was observed to be the most bioactive fraction. It was shown to possess effective cytotoxic and cell cycle inhibitory activities against SCC-9 cells. The hexane fraction also induced apoptosis in SCC-9 cells which was further established at the level of caspase 3 and 8 gene expressions. Conclusion: Overall, the results clearly establish the potential of ICLH extract to inhibit cell proliferation and induce apoptosis in the SCC-9 cells. Further analysis of the ICLH fraction could result in development of effective anticancer therapeutics. The natural abundance of I. cylindrica with its wide geographic distribution could make it a preferred natural resource for obtaining novel, cost-effective, anticancer therapeutics with minimal systemic side effects.     

Hexane extracts of garlic cloves induce apoptosis through the generation of reactive oxygen species in Hep3B human hepatocarcinoma cells

Garlic (Allium sativum) compounds have recently received increasing attention due to their cancer chemopreventive properties, and their anticancer activities are extensively reported in many cancer cell lines. However, the anticancer activity and the signaling pathway associated with the induction of apoptosis by extracts of garlic cloves have not been elucidated. In this study, we examined the effects of hexane extracts of garlic cloves (HEGCs) on reactive oxygen species (ROS) production and the association of these effects with apoptotic cell death, using a Hep3B human hepatocarcinoma cell line in?vitro. The results demonstrated that HEGCs mediate ROS production, and that this mediation is followed by a collapse of mitochondrial membrane potential (MMP, ΔΨm), the downregulation of anti-apoptotic Bcl-2 and Bcl-xL and the activation of caspase-9 and -3. HEGCs also promoted the activation of caspase-8 and the downregulation of Bid, a BH3-only pro-apoptotic member of the Bcl-2. However, the apoptotic phenomena displayed by HEGCs were significantly diminished by the presence of z-VAD-fmk (non-selective caspase inhibitor). Moreover, N-acetyl-L-cysteine (NAC), a widely used ROS scavenger, effectively blocked the HEGC-induced apoptotic effects via the inhibition of ROS production and MMP collapse. These observations clearly indicate that HEGC-induced ROS are key mediators of MMP collapse, which leads to the induction of apoptosis, followed by caspase activation.     

Induction of apoptosis and autophagic cell death by the vanillin derivative 6-bromine-5-hydroxy-4-methoxybenzaldehyde is accompanied by the cleavage of DNA-PKcs and rapid destruction of c-Myc oncoprotein in HepG2 cells

Autophagy is a regulated lysosomal pathway involving the bulk degradation of cytoplasmic contents, and is an emerging attractive therapeutic approach for treating cancers. In the present study, we demonstrates that bromovanin (6-bromine-5-hydroxy-4-methoxybenzaldehyde), a vanillin derivative, exhibits a potent antiproliferative effect on a broad spectrum of cancer cell lines, but it induces apoptosis with a large variation in extent on different cancer cell lines. Ultrastructural observation in transmission electron microscopy reveals that autophagy is another type of cell death induced by bromovanin in HepG2 cells. Treatment with bromovanin significantly increases cellular ROS level as well as elicits DNA double-strand breaks as indicated by comet assay and the increased phosphorylated H2AX. Cleavage and inactivation of DNA-PKcs induced by bromovanin is found to occur concurrently with a rapid destruction of c-Myc oncoprotein. These multiple effects of bromovanin, especially the induction of both apoptosis and autophagy, make it very appealing for the development as a novel anticancer drug.     

Antiproliferative Activity of the Methanolic Extract of Withania Somnifera Leaves from Faifa Mountains,Southwest Saudi Arabia,against Several Human Cancer Cell Lines

Cancer represent one of the most serious health problems and major causes of death around the world. Many anticancer drugs in clinical use today are natural products or derived from natural sources. Withania somnifera (L.) Dunal is a small shrub widely distributed in many parts of the world including Saudi Arabia. The antiproliferative activities of the methanolic extract of W. somnifera leaves collected from Faifa mountains, southwest Saudi Arabia against MCF-7, HCT116 and HepH2 cell lines were investigated. The extract showed a strong antiproliferative activity against all cell lines with IC50 values of 3.35, 2.19 and 1.89 g/ml, respectively. Flow cytometry results showed that the extract arrested the cell cycle at S phase, and the increase in the caspase 3 activity suggested that the extract could induce cell apoptosis by a caspase mediated pathway. These results demonstrated that the methanolic extract of W. somnifera leaves collected from Faifa mountains has comparable strong antiproliferative activities to samples collected from different locations.     

Bevacizumab and CCR2 Inhibitor Nanoparticles Induce Cytotoxicity-Mediated Apoptosis in Doxorubicin-Treated Hepatic and Non-Small Lung Cancer Cells

Non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC) are very common in certain populationaround the world. Despite the recent advances in their diagnosis and therapy, their prognosis remains poor due to thedevelopment resistance to drug. Although doxorubicin (DOX) is considered to be one of the most anti-solid tumordrugs, developed resistance is contributing to unsuccessful outcome. The rationale of the current study is to explorethe sensitizing capability of the DOX-treated cancer cells using the anticancer agents; bevacizumab (avastin; AV) andCCR2 inhibitor (CR) in their free- and nano-formulations. Here, the average size, polydispersity index (PDI), zetapotential, and entrpment effeciency (EE%) of the synthesized nanoparticles were measured. We investigated the effectof these platforms on the proliferation, apoptosis, necrosis, nitric oxide (NO), malondialdehyde (MDA), and zinc levelsof human HCC (HepG2 and Huh-7) and NSCLC (A549) cancer cell lines. Glucose consumption rates using Huh-7and A549 cancer cells were tested upon treatments. We demonstrated that AV and CR nano-treatments significantlysuppressed A549 cell viability and activated apoptosis by NO level elevation. We concluded that AVCR NP plusDOX significantly induces A549 cytotoxicity-mediated apoptosis more than Huh-7 and HepG2 cells. This drug-drugnano-combination induced Huh-7 cytotoxicity-mediated apoptosis more than HepG2 cells. In conclusion, AVCR NPsensitized DOX-treated A549 and Huh-7 cells through reactive oxygen species (ROS)-stimulated apoptosis. Takentogether, our data suggested that the CR plus AV nano-platforms would be a potential personalized medicine-basedstrategy for treating CCR2-positive NSCLC and HCC patients in the near future.     

Anti-proliferative activity of Bupleurum scrozonerifolium in A549 human lung cancer cells in vitro and in vivo

Nan-Chai-Hu, the root of Bupleurum scorzonerifolium, is a traditional Chinese herb used in treatment of liver diseases such as hepatitis and cirrhosis. We recently reported that the acetone extract of B. scorzonerifolium (BS-AE) could inhibit proliferation and induce apoptosis in A549 human lung cancer cells. We further examined its anti-proliferative mechanisms and in vivo anticancer effect. Our results showed that BS-AE had the ability to cause cell cycle arrest in G2/M phase, inducing tubulin polymerization, and activating caspase-3 and -9 in A549 cells. BS-AE-induced apoptosis could be blocked by the broad caspase inhibitor z-VAD-fmk in majority. The result of in vivo study showed that BS-AE could suppress growth in A549 subcutaneous xenograft tumors. These results indicate that BS-AE exerts antiproliferative effects on A549 cells in vitro and in vivo, and prompted us to further evaluate and elucidate the chemical composition profile of BS-AE.     

Deactivation of cisplatin-resistant human lung/ovary cancer cells with pyropheophorbide-α methyl ester-photodynamic therapy

The aim of this study was to determine whether photodynamic therapy (PDT) alone or combined with cisplatin (DDP), can deactivate cisplatin-resistant cancer cells. Human cancer cell lines A549 and SKOV3, and chemoresistant sublines A549/DDP and SKOV3/DDP, were subjected to PDT, DDP, or PDT combined with DDP. Cell viability and apoptosis were analyzed, and then intracellular reactive oxygen species (ROS) and proteins related to apoptosis were determined. PDT caused cell death, and PDT combined with DDP led to the highest percentage of dead cells in 4 cell lines; similar results were detected in ROS; a quantification evaluation manifested that the combined effect was addition. DDP increased the percentage of apoptotic cells, and the ROS level in A549 and SKOV3 cells, which was not observed in A549/DDP and SKOV3/DDP cells. Western blot revealed an increase of caspase 3 and Bax, and a decrease of Bcl-2, demonstrating the occurrence of apoptosis. The data suggest that PDT can efficiently deactivate resistant cells and enhance the action of DDP against resistant cancer cells.     

Polyphenol Content,Antioxidant, Cytotoxic,and Genotoxic Activities of Bombax ceiba Flowers in Liver Cancer Cells Huh7

Objective: Bombax ceiba (red Silk cotton tree) has great ethnopharmacological significance due to its widespread use to treat various diseases such as dysentery, inflammation, and tuberculosis. Despite decades of research, the studies on the in vitro anticancer/genotoxic activity of B. ceiba flower remains restricted. Thus, the present research explored the effect of ethanol extract from B. ceiba flowers on three human cancer cells, including lung A549 and liver HepG2 and Huh7 cell lines. Methods: Cytotoxic and genotoxic activity of B. ceiba extract was examined by MTT and comet assay, respectively. Further, B. ceiba extract was analysed to determine total polyphenol content and DPPH antiradical scavenging activity. Results: ethanol extract from B. ceiba flowers had a high polyphenols content with very potent antioxidant activity. Further, B. ceiba extract displayed moderate cytotoxicity against Huh7 cells and no cytotoxicity against HepG2 and A549 cells. The comet assay findings showed that Huh7 cells treated with four concentrations of B. ceiba extract (¼ IC50, ½ IC50, IC50, and double IC50) increased the comet tail formation within 48 h in a concentration-dependent manner. Conclusion: ethanol extract from B. ceiba flowers exhibited its cytotoxicity through induction of DNA fragmentation in Huh7 cells.     

Cytotoxicity Assessment of Six Different Extracts of Abelia triflora leaves on A-549 Human Lung Adenocarcinoma Cells

The present investigation was designed to assess the anticancer activity of six different leaf extracts (ethylacetate, methanol, chloroform, petroleum ether, n-butanol, and water soluble) of Abelia triflora on A-549 humanlung adenocarcinoma epithelial cells. A-549 cells were exposed to 10-1000 μg/ml concentrations of the leaf extractsof A. triflorafor 24 h and then percentage cell viability was assessed by 3-(4,5-dimethylthiazol-2yl)-2,5-biphenyltetrazolium bromide (MTT) assay. The results showed that leaf extracts of A. triflora significantly reduced theviability of A-549 cells in a concentration-dependent manner. Decrease was recorded as 31% with ethyl acetate,36% with methanol, 46% with chloroform, 54% with petroleum ether, 62% with n-butanol, and 63% withwater soluble extracts at 1000 μg/ml each. Among the various plant extracts, ethyl acetate extract showed thehighest decrease in the percentage cell viability, followed by methanol, chloroform, petroleum ether, n-butanol,and water soluble extracts. Our results demonstrated preliminary screening of anticancer activity of differentsoluble extracts of A. triflora extracts against A-549 cells, which can be further used for the development of apotential therapeutic anticancer agents.     

洛铂对鼻咽癌抗肿瘤作用的体外实验研究

目的 探讨洛铂对鼻咽癌的体外抗肿瘤作用及其可能的机制,为洛铂用于鼻咽癌的临床治疗提供实验依据。方法 在体外将不同浓度洛铂分别作用于低分化鼻咽癌细胞株CNE2、HONE1和SUNE1,采用CCK-8法检测细胞活性;碘化丙啶（PI）染色分析细胞中DNA的含量,流式细胞仪检测细胞周期情况;采用Annexin V-FITC／PI双染法检测细胞凋亡情况;采用蛋白印迹法（Western blotting）检测细胞周期和细胞凋亡相关蛋白的表达。结果 在体外洛铂作用3种鼻咽癌细胞株48h具有明显的细胞抑制作用,并表现出浓度依赖性。洛铂作用鼻咽癌细胞株CNE2、HONE1、SUNE1的半数抑制浓度（IC₅₀）分别为（4.05±0.49）μmol/L、（4.32±1.17）μmol/L和（2.51±0.15）μmol／L。洛铂作用3种鼻咽癌细胞株48h后,在较低浓度（0.125倍IC₅₀）时将细胞周期阻滞在G₂期,同时伴随G₂期相关蛋白Cyclin B1和phospho-cdc2（Tyr15）表达增高;而在较高浓度（0.5倍IC₅₀）时则诱导细胞呈Caspase依赖性细胞凋亡,并具有浓度依赖性。结论 洛铂对鼻咽癌细胞具有明显的细胞毒作用,且呈浓度依赖性;其机制表现为双重作用,即在较低浓度时阻滞细胞于G₂期和在较高浓度时诱导细胞凋亡,这为洛铂用于鼻咽癌的临床治疗提供可能性。     

Evaluation of cell death caused by an ethanolic extract of Serenoae repentis fructus (Prostasan) on human carcinoma cell lines

BACKGROUND: Phytotherapy is a third approach for treating lower urinary tract symptoms associated with benign prostatic hyperplasia (BPH). The lipido-sterolic extract of the fruit of Serenoa repens is one of the more widely used phytotherapeutic agents in this regard. MATERIALS AND METHODS: The effect of an ethanolic extract of S. repens (10-1000 microg/ml) was tested in hormone-sensitive LNCaP, MCF-7 and hormone-insensitive DU 145, MDA MB231 prostate, breast carcinoma cell lines, renal Caki-1, urinary bladder J82, colon HCT 116 and lung A 549 cancer cells. Its cell growth inhibitory and apoptosis-inducing effects were tested using WST-1 assay and flow cytometry (Annexin V/PI stain) and/or by colorimetric assay (APOPercentage assay). RESULTS: The S. repens extract induced a dose-dependent antiproliferative effect on all human malignant cells tested, with GI50 values between 107 and 327 pmicro/ml. In hormone-sensitive prostate LNCaP and breast MCF-7 cell lines, the effect of extract expressed in GI50 was 2.2- and 2.5-fold more potent (p < 0.01) than in hormone-insensitive DU 145 and MDA MB231 cells. The proportion of apoptotic cells, except in A549 cells, lay between 22.5-36.3%. S. repens extract did not induce apoptosis in lung cancer A 549 cells. CONCLUSION: This study showed that the antiproliferative effect exerted by the ethanolic extract of S. repens is at least triggered by induction of apoptosis. These in vitro data provide some information that may be useful for clinical use and render S. repens extract an interesting tool for new applications.     

Bioassay-Guided Isolation and Evaluation of Antiproliferative Effects of (Z)-Ethylidene-4, 6-Dimethoxycoumaran-3-One from Pogostemon Quadrifolius (Benth.)

The aim of the study was to isolate and identify the major cytotoxic principle from plant leaves of Pogostemon quadrifolius (Benth.) and evaluate its antiproliferative potential against human cancer cells. Plant leaves were extracted sequentially with a soxhlet apparatus, using petroleum ether, chloroform and methanol solvents. Petroleum ether and chloroform extracts exhibited antiproliferative properties against Caco-2, HeLa, THP-1, MCF-7 and Jurkat E6-1cancer cell lines tested, but methanol extracts failed to exhibit such activity. The major antiproliferative principle from petroleum ether and chloroform extracts was isolated with the help of bioassay guided column chromatography. This cytotoxic compound was further analysed by UV, TLC, HPLC, LC-MS, GC-MS and NMR analyses and was identified to be novel: (Z)-ethylidene-4,6-dimethoxycoumaran-3-one (Compound 1). The half-maximal inhibitory concentrations for proliferation (IC50) exhibited by compound 1 were 19.4, 23.1, 22.1, 35.9 and 8.32 μM against Caco-2, HeLa, THP-1, MCF-7 and Jurkat E6-1 cancer cell lines, respectively. Further experiments revealed that compound 1 triggered the apoptosis mode of cell death in cancer cell lines. Thus, the present study allowed isolation and identification of a novel cytotoxic natural compound, (Z)-ethylidene-4,6-dimethoxycoumaran-3-one, from plant leaves of P. quadrifolius (Benth.). Our pre-clinical study also indicated that compound 1 is particularly active in the acute T cell leukemia cell line (Jurkat E6-1) with potential for application as a chemotherapeutic agent in the future.     

Apoptotic signaling in bufalin‐ and cinobufagin‐treated androgen‐dependent and ‐independent human prostate cancer cells

Prostate cancer has its highest incidence in the USA and is becoming a major concern in Asian countries. Bufadienolides are extracts of toxic glands from toads and are used as anticancer agents, mainly on leukemia cells. In the present study, the antiproliferative and apoptotic mechanisms of bufalin and cinobufagin on prostate cancer cells were investigated. Proliferation of LNCaP, DU145, and PC3 cells was measured by 3‐(4,5‐dimethylthiazol‐2‐yle)‐2,5‐diphenyltetrazolium bromide assay and the doubling time (tD) was calculated. Bufalin and cinobufagin caused changes in the tD of three prostate cancer cell lines, which were more significant than that of human mesangial cells. In addition, bufadienolides induced prostate cancer cell apoptosis more significantly than that in breast epithelial cell lines. After treatment, the caspase‐3 activity and protein expression of caspase‐3, ‐8, and ‐9 were elevated. The expression of other apoptotic modulators, including mitochondrial Bax and cytosolic cytochrome c, were also increased. However, expression of p53 was only enhanced in LNCaP cells. Downregulation of p53 by antisense TP53 restored the cell viability suppressed by bufalienolides. Furthermore, the increased expression of Fas was more significant in DU145 and PC3 cells with mutant p53 than in LNCaP cells. Transfection of Fas small interfering RNA restored cell viability in the bufadienolide‐treated cells. These results suggest that bufalin and cinobufagin suppress cell proliferation and cause apoptosis in prostate cancer cells via a sequence of apoptotic modulators, including Bax, cytochrome c, and caspases. The upstream mediators might be p53 and Fas in androgen‐dependent LNCaP cells and Fas in androgen‐independent DU145 and PC3 cells. (Cancer Sci 2008; 99: 2467–2476)     

Portulaca oleracea Seed Oil Exerts Cytotoxic Effects on Human Liver Cancer (HepG2) and Human Lung Cancer (A-549) Cell Lines

Portulaca oleracea (Family: Portulacaceae), is well known for its anti-inflammatory, antioxidative, antibacterial,and anti-tumor activities. However, cytotoxic effects of seed oil of Portulaca oleracea against humanliver cancer (HepG2) and human lung cancer (A-549) cell lines have not been studied previously. Therefore, thepresent study was designed to investigate the cytotoxic effects of Portulaca oleracea seed oil on HepG2 and A-549cell lines. Both cell lines were exposed to various concentrations of Portulaca oleracea seed oil for 24h. After theexposure, percentage cell viability was studied by (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)(MTT), neutral red uptake (NRU) assays, and cellular morphology by phase contrast inverted microscopy. Theresults showed a concentration-dependent significant reduction in the percentage cell viability and an alteration inthe cellular morphology of HepG2 and A-549 cells. The percentage cell viability was recorded as 73%, 63%, and54% by MTT assay and 76%, 61%, and 50% by NRU assay at 250, 500, and 1000 μg/ml, respectively in HepG2cells. Percentage cell viability was recorded as 82%, 72%, and 64% by MTT assay and 83%, 68%, and 56%by NRU assay at 250, 500, and 1000 μg/ml, respectively in A-549 cells. The 100 μg/ml and lower concentrationswere found to be non cytotoxic to A-549 cells, whereas decrease of 14% and 12% were recorded by MTT andNRU assay, respectively in HepG2 cells. Both HepG2 and A-549 cell lines exposed to 250, 500, and 1000 μg/ml of Portulaca oleracea seed oil lost their normal morphology, cell adhesion capacity, become rounded, andappeared smaller in size. The data from this study showed that exposure to seed oil of Portulaca oleracea resultedin significant cytotoxicity and inhibition of growth of the human liver cancer (HepG2) and human lung cancer(A-549) cell lines.     

Anticancer and Antioxidant Activities of Aqueous and Ethanolic Bark Extracts of <i>Acer Tegmentosum</i> Maxim (Aceaceae) on Tumor Cell Lines

The medicinal plant of Acer tegmentosum Maxim is traditionally used in the southern part of Asia to treat oxidative stress-related diseases, including cancer, diabetes mellitus , wounds, infections, etc. Based on this, the current study was designed to investigate the phytochemical analysis, antioxidants and anti-cancer activities of Acer tegmentosum Maxim (AT). The total phenolic content (TPC), total flavonoid content (TFC), free radicals scavenging (DPPH and ABTS), chemical constituents as well as cytotoxicity potential ATWE (Acer tegmentosum Maxim water extracts) and ATEE (Acer tegmentosum Maxim ethanolic extracts) were tested. The cytotoxic efficacy ATWE and ATEE were studied in Human embryonic kidney 293 cells (HEK 293), human lung cancer cell lines (A549), prostate cancer cells (PC3) and breast cancer cells (MDA-MB 231). The results revealed that, TPC ranged between in 199.97 ± 0.09 mg GAR/g extract in ATWE and 103.48 ± 0.82 mg GAR/g extract in ATEE, TFC were 72.10 ± 0.07 mg RE/g extract in ATWE, and 47.28 ± 0.55 mg RE/g extract in ATEE. Aside it showed a promising antioxidant scavenging activity against DPPH and ABTS radicals. The antioxidant capacity of the two extracts increased in a dose-dependent manner. ATEE and ATWE had little difference in the scavenging rate of DPPH free radicals, and its radical scavenging activity were reached about 70% at 1000 μg/mL. ATWE had significant cytotoxicity to all the tested cancer cell lines of A549, PC3, and MDA-MB 231. The anti-cancer activity of ATWE was better than ATEE, and the IC₅₀ value for A549 cells, PC3 cells, and MDA-MB cells were 96.32 ± 5.96, 198.58 ± 10.35 and 365.27 ± 19.72 μg/mL, respectively. The fluorescent staining (AO/EB, PI, Rh123, ROS) studies explored that ATWE could target the cancer cell via nuclear damage, excessive production of ROS and loss of mitochondrial membrane potential, suggesting that activation of endogenous apoptosis pathways. These results proved that AT extracts (especially ATWE) had significant antioxidants and anti-cancer activities, implying a possible pharmacological application of AT.     

Decitabine, a DNA methyltransferase inhibitor, induces apoptosis in human leukemia cells through intracellular reactive oxygen species generation

The DNA methyltransferase inhibitor decitabine, 5-aza-2'-deoxycytidine, has been found to exert anti-metabolic and anticancer activities when tested against various cultured cancer cells. Furthermore, decitabine has been found to play critical roles in cell cycle arrest and apoptosis in various cancer cell lines; however, these roles are not well understood. In this study, we investigated decitabine for its potential anti-proliferative and apoptotic effects in human leukemia cell lines U937 and HL60. Our results indicated that treatment with decitabine resulted in significantly inhibited cell growth in a concentration- and time-dependent manner by the induction of apoptosis. Decitabine-induced apoptosis in U937 and HL60 cells was correlated with the downregulation of anti-apoptotic Bcl-2, XIAP, cIAP-1 and cIAP-2 protein levels, the cleavage of Bid proteins, the activation of caspases and the collapse of mitochondrial membrane potential (MMP). However, apoptosis induced by decitabine was attenuated by caspase inhibitors, indicating an important role for caspases in decitabine responses. The data further demonstrated that decitabine increased intracellular reactive oxygen species (ROS) generation. Moreover, N-acetyl-L-cysteine, a widely used ROS scavenger, effectively blocked the decitabine-induced apoptotic effects via inhibition of ROS production and MMP collapse. These observations clearly indicate that decitabine-induced ROS in human leukemia cells are key mediators of MMP collapse, which leads to apoptosis induction followed by caspase activation.     

Doxorubicin-induced apoptosis and chemosensitivity in hepatoma cell lines

PURPOSE: Doxorubicin (DOX) is a commonly used anticancer drug which causes DNA damage and kills cancer cells mainly by apoptosis. However, the process leading to killing of cancer cells and the molecular basis of resistance to DOX are not well understood. To evaluate the role of p53 and the cellular effects of DOX on hepatoma cell lines, we examined three hepatoma cell lines with different p53 status--Huh-7 (mutated p53), HepG2 (wild-type p53) and Hep3B (deleted p53). METHODS: The chemosensitivity of the three hepatoma cell lines was assessed using the MTT assay, and cell cycle distribution was evaluated by flow cytometry. Western blotting and immunostaining were employed to examine the protein alterations in response to DOX treatment, and a DNA fragmentation assay was performed to detect apoptosis. RESULTS: Of the three cell lines, HepG2 was found to be most resistant to DOX, followed by Hep3B, and Huh-7 was most sensitive to DOX treatment. HepG2 showed G1 arrest 24 h after drug administration and upregulation of p53 protein level in a time-dependent manner. In Hep3B cells (deleted p53), G2/M phase arrest was observed soon after drug administration, accompanied by induced apoptosis that was p53-independent. In Huh-7 cells (mutated p53), which were most sensitive to DOX, there was neither G1 nor G2 arrest, and the level of p53 mutated protein was downregulated after DOX treatment. MDM2 and p27 proteins were downregulated in all cell lines independently of p53 status. p21 was upregulated following p53 activation at low doses of DOX in HepG2 cells, but at higher doses, p21 was downregulated in Huh-7 and HepG2 cells. CONCLUSION: DOX confers different chemosensitivity on different hepatoma cell lines with different p53 status. The contrasting relationships between chemosensitivity and p53 status and expression suggest that DOX-induced apoptosis and cell death involve pathways that are independent of p53.