Caspase-dependent Apoptotic Pathway Research Articles

An Sp1 transcription factor coordinates caspase-dependent and -independent apoptotic pathways.

During animal development, the proper regulation of apoptosis requires the precise spatial and temporal execution of cell-death programs, which can include both caspase-dependent and caspase-independent pathways1, 2. While the mechanisms of caspase-dependent and caspase-independent cell killing have been examined extensively, how these pathways are coordinated within a single cell that is fated to die is unknown. Here we show that the C. elegans Sp1 transcription factor SPTF-3 specifies the programmed cell deaths of at least two cells, the sisters of the pharyngeal M4 motor neuron and of the AQR sensory neuron, by transcriptionally activating both caspase-dependent and caspase-independent apoptotic pathways. SPTF-3 directly drives the transcription of the gene egl-1, which encodes a BH3-only protein that promotes apoptosis through the activation of the CED-3 caspase3. In addition, SPTF-3 directly drives the transcription of the AMPK-related gene pig-1, which encodes a protein kinase and functions in apoptosis of the M4 sister and AQR sister independently of the pathway that activates CED-34, 5. Thus, a single transcription factor controls two distinct cell-killing programs that act in parallel to drive apoptosis. Our findings reveal a bivalent regulatory node for caspase-dependent and caspase-independent pathways in the regulation of cell-type specific apoptosis. We propose that such nodes might act in a general mechanism for regulating cell-type specific apoptosis and could define therapeutic targets for diseases involving the dysregulation of apoptosis through multiple cell-killing mechanisms.

Pyrimethamine sensitizes pituitary adenomas cells to temozolomide through cathepsin B-dependent and caspase-dependent apoptotic pathways

Invasive pituitary adenomas (PAs) are generally refractory to conventional therapy and salvage treatment with temozolomide (TMZ). In addition to antiprotozoan effects, pyrimethamine (PYR) has recently shown its strong antitumor activity as an antineoplastic agent or in combination with TMZ in metastatic melanoma cells. In this study, the effects of TMZ, PYR or TMZ/PYR combination on rat/mouse PA cell lines αT3-1, GH3, MMQ and ATt-20 as well as GH3 xenograft tumor model were evaluated. TMZ/PYR combination synergistically inhibited proliferation, invasion and induced apoptosis of these PA cell lines in vitro. Strikingly, combination treatment with TMZ and PYR produced synergistic antitumor activity and enhanced the survival rate of GH3 xenograft tumor models without increasing systemic side effects. In addition, TMZ/PYR induced cell cycle arrest, increased DNA damage, upregulated the expression of cathepsin B, BAX, cleaved PARP and phosphorylated histone H2AX as well as elevated caspase3/7, 8 and 9 activities. The decreased expression of Bcl-2, MMP-2 and MMP-9 alone with cytochrome c release from mitochondria into the cytosol was also observed in the TMZ/PYR combination group. The increase in cell apoptosis due to combination with PYR was rescued by leucovorin. These data suggest that PYR may enhance the efficacy of TMZ via triggering both cathepsin B-dependent and caspase-dependent apoptotic pathways. Therefore, combination of PYR and TMZ may provide a novel regimen for invasive PAs refractory to standard therapy and TMZ.

Abstract 4866: CLEFMA induces apoptosis and downregulates inflammatory markers in lung cancer model.

Abstract Background: Inflammation is a precursor for many cancers. NF-kB plays an important role in inflammation, is associated with cancerous growth and has been identified as a potential target for development of anticancer therapies. Lung cancer is a classic example of inflammation-induced carcinogenesis where it prevents apoptosis and stimulates production of pro-inflammatory cytokines. CLEFMA or 4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid] is a new molecule that potently inhibits proliferation of various cancer cells without affecting normal cells. In this study, we evaluated the antiproliferative activity of CLEFMA in a mouse xenograft model of human lung cancer. Methods: Cell viability was determined in human lung cancer cells H441 and A549 after treatment with CLEFMA. Immunobloting studies were performed in cell lysates and tissue homogenates for anti-apoptotic and pro-apoptotic markers. These were further confirmed by immunohistochemical studies. The ability of CLEFMA in reducing the metabolic turnover in tumor tissue was examined by positron emission tomography using F-18-FDG as a biomarker of glucose metabolism. Results: We found that CLEFMA-induced suppression of lung adenocarcinoma H441 and A549 cell proliferation was associated with cleavage of caspases 3/9 and PARP, suggesting apoptotic cell death. In vivo, CLEFMA demonstrated a dose-dependent suppression of tumor growth associated with reduction of tumor proliferation marker Ki-67. Non-invasive PET imaging with F-18-flurodeoxyglucose also showed abrogation of glucose uptake in tumor tissue. In the tumor tissue homogenates, we found inhibition of anti-apoptotic markers cIAP1, Bcl-XL, BCL2 and survivin, and upregulation of pro-apoptotic BID cleavage, BAX and PARP. Further, CLEFMA inhibited NF-kB at mRNA level and reduced phospho-p65 translocation into the nucleus. It also reduced the expression of COX-2 in tumor tissue, and significantly reduced serum pro-inflammatory cytokines TNF-α and IL-6. Conclusions: The findings suggest that CLEFMA inhibits growth of lung cancer xenografts by activating caspase-dependent apoptotic pathway, and the tumor suppression is associated with anti-inflammatory and anti-metastatic effects, perhaps mediated by NF-kB. Citation Format: Vivek R. Yadav, Kaustuv Sahoo, Vibhudutta Awasthi. CLEFMA induces apoptosis and downregulates inflammatory markers in lung cancer model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4866. doi:10.1158/1538-7445.AM2013-4866

Induction of Cell Cycle Arrest and Apoptosis in Human Osteosarcoma U-2 OS Cells by Solanum lyratum Extracts

This research focused on a Chinese herb medicine, Solanum lyratum Thunb (Solanaceae) by ethanol extracts (SLE) for investigating the molecular anticancer mechanism in vitro for exploring the means of cell death through the effects on mitochondrial function. We found that SLE induced cytotoxic effects in human osteosacroma U-2 OS cells, and these effects include cell morphological changes, a decrease of the percentage of viable cells and induction of apoptosis. The results suggest that cell death induced by SLE is closely related to apoptosis based on the observations of DAPI staining and sub-G1 phase in U-2 OS cells. Flow cytometric assays also showed that SLE promoted the production of reactive oxygen species and nitric oxide but decreased the levels of mitochondrial membrane potential and promoted the activations of caspase-8 and -9 in U-2 OS cells. SLE inhibited the level of Bcl-2 but promoted the Bax level, and both proteins led to the release of cytochrome c from mitochondria to cytosol and activation of caspase-9 and -3, resulting in the apoptotic death which is mediated through the mitochondrial pathway. Taken together, SLE was demonstrated to be effective in killing U-2 OS osteosacroma cells via the ROS-promoted and mitochondria- and caspase-dependent apoptotic pathways.

Hydrogen peroxide induces cell death in human TRAIL-resistant melanoma through intracellular superoxide generation

Intracellular reactive oxygen species (ROS) such as hydrogen peroxide (H(2)O2()) are thought to mediate apoptosis induced by death receptor ligands, including tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, the role of H(2)O(2) is controversial, since some evidence suggests that H(2)O(2) acts as an anti-apoptotic factor. Here, we show that exogenously applied H(2)O(2) (30-100 µM) induces cell death in TRAIL-resistant human melanoma cells via intracellular superoxide (O(2)-) generation. H(2)O(2) induced apoptotic or necrotic cell death, depending on the concentration of the oxidant applied; low concentrations of H(2)O(2) preferentially activated the caspase-dependent apoptotic pathway, while high concentrations of H(2)O(2) induced apoptotic and necrotic cell death in a caspase-independent manner. The H(2)O(2)-induced cell death was associated with increased mitochondrial membrane potential collapse and caspase-3/7 activation and ER stress responses including caspase-12 and X-box-binding protein-1 (XBP-1) activation. H(2)O(2) induced intracellular O2- generation even within the mitochondria, while TRAIL did not. The superoxide dismutase mimetic antioxidant MnTBaP [Mn (III) tetrakis (4-benzonic acid) porphyrin chloride] inhibited the H(2)O(2)-induced O(2)- generation, apoptosis and XBP-1 and caspase-12 activation at comparable concentrations. Importantly, H(2)O(2) treatment caused minimal O(2)- generation and apoptosis in normal primary melanocytes. These data show that H(2)O(2) induces endoplasmic reticulum-associated cell death via intracellular O(2)- generation and that malignant melanoma cells are more susceptible than normal cells to this oxidative cell death. The findings suggest that H(2)O(2) has therapeutic potential in the treatment of TRAIL-resistant melanoma.

Downregulation of Fas activity rescues early onset of diabetes inc-KitWv/+mice

c-Kit and its ligand stem cell factor (SCF) are important for β-cell survival and maturation; meanwhile, interactions between the Fas receptor (Fas) and Fas ligand are capable of triggering β-cell apoptosis. Disruption of c-Kit signaling leads to severe loss of β-cell mass and function with upregulation of Fas expression in c-Kit(Wv/+) mouse islets, suggesting that there is a critical balance between c-Kit and Fas activation in β-cells. In the present study, we investigated the interrelationship between c-Kit and Fas activation that mediates β-cell survival and function. We generated double mutant, c-Kit(Wv/+);Fas(lpr/lpr) (Wv(-/-)), mice to study the physiological and functional role of Fas with respect to β-cell function in c-Kit(Wv/+) mice. Isolated islets from these mice and the INS-1 cell line were used. We observed that islets in c-Kit(Wv/+) mice showed a significant increase in β-cell apoptosis along with upregulated p53 and Fas expression. These results were verified in vitro in INS-1 cells treated with SCF or c-Kit siRNA combined with a p53 inhibitor and Fas siRNA. In vivo, Wv(-/-) mice displayed improved β-cell function, with significantly enhanced insulin secretion and increased β-cell mass and proliferation compared with Wv(+/+) mice. This improvement was associated with downregulation of the Fas-mediated caspase-dependent apoptotic pathway and upregulation of the cFlip/NF-κB pathway. These findings demonstrate that a balance between the c-Kit and Fas signaling pathways is critical in the regulation of β-cell survival and function.

Increase in antioxidant and anticancer effects of ginsenoside Re–lysine mixture by Maillard reaction

Ginsenosides are the main active components of Panax ginseng. Structural changes in diol type ginsenosides along with generation of Maillard reaction products (MRPs) are strongly associated with increased free radical-scavenging activities. Ginsenoside Re, one of the major triol type ginsenosides of P. ginseng, possesses a hydrophobic four-ring steroid-like structure with hydrophilic sugar moieties at carbons-3 and -20. The aim of the present study was to identify changes in the structure, antioxidant and anticancer effects of ginsenoside Re upon Maillard reaction. Ginsenoside Re was transformed into less-polar ginsenosides, namely Rg2, Rg6 and F4 by heat-processing. Free radical-scavenging activity of the ginsenoside Re–lysine mixture increased upon heat processing. This improved free radical-scavenging activity mediated by antioxidant MRPs, which were generated through Maillard reaction of a glucosyl moiety separated from carbon-20 of ginsenoside Re and lysine. The increased anticancer effect of ginsenoside Re–lysine mixture upon heat processing was mainly derived from the generation of less-polar ginsenosides through the regulation of Bcl-2 and Bax, as well as caspase-dependent apoptotic pathway. These results reported here have shed significant new lights on the mechanism of increased antioxidant and anticancer effects of P. ginseng upon heat processing.

Frankincense essential oil prepared from hydrodistillation of Boswellia sacra gum resins induces human pancreatic cancer cell death in cultures and in a xenograft murine model

BackgroundRegardless of the availability of therapeutic options, the overall 5-year survival for patients diagnosed with pancreatic cancer remains less than 5%. Gum resins from Boswellia species, also known as frankincense, have been used as a major ingredient in Ayurvedic and Chinese medicine to treat a variety of health-related conditions. Both frankincense chemical extracts and essential oil prepared from Boswellia species gum resins exhibit anti-neoplastic activity, and have been investigated as potential anti-cancer agents. The goals of this study are to identify optimal condition for preparing frankincense essential oil that possesses potent anti-tumor activity, and to evaluate the activity in both cultured human pancreatic cancer cells and a xenograft mouse cancer model.MethodsBoswellia sacra gum resins were hydrodistilled at 78°C; and essential oil distillate fractions were collected at different durations (Fraction I at 0–2 h, Fraction II at 8–10 h, and Fraction III at 11–12 h). Hydrodistillation of the second half of gum resins was performed at 100°C; and distillate was collected at 11–12 h (Fraction IV). Chemical compositions were identified by gas chromatography–mass spectrometry (GC-MS); and total boswellic acids contents were quantified by high-performance liquid chromatography (HPLC). Frankincense essential oil-modulated pancreatic tumor cell viability and cytotoxicity were determined by colorimetric assays. Levels of apoptotic markers, signaling molecules, and cell cycle regulators expression were characterized by Western blot analysis. A heterotopic (subcutaneous) human pancreatic cancer xenograft nude mouse model was used to evaluate anti-tumor capability of Fraction IV frankincense essential oil in vivo. Frankincense essential oil-induced tumor cytostatic and cytotoxic activities in animals were assessed by immunohistochemistry.ResultsLonger duration and higher temperature hydrodistillation produced more abundant high molecular weight compounds, including boswellic acids, in frankincense essential oil fraactions. Human pancreatic cancer cells were sensitive to Fractions III and IV (containing higher molecular weight compounds) treatment with suppressed cell viability and increased cell death. Essential oil activated the caspase-dependent apoptotic pathway, induced a rapid and transient activation of Akt and Erk1/2, and suppressed levels of cyclin D1 cdk4 expression in cultured pancreatic cancer cells. In addition, Boswellia sacra essential oil Fraction IV exhibited anti-proliferative and pro-apoptotic activities against pancreatic tumors in the heterotopic xenograft mouse model.ConclusionAll fractions of frankincense essential oil from Boswellia sacra are capable of suppressing viability and inducing apoptosis of a panel of human pancreatic cancer cell lines. Potency of essential oil-suppressed tumor cell viability may be associated with the greater abundance of high molecular weight compounds in Fractions III and IV. Although chemical component(s) responsible for tumor cell cytotoxicity remains undefined, crude essential oil prepared from hydrodistillation of Boswellia sacra gum resins might be a useful alternative therapeutic agent for treating patients with pancreatic adenocarcinoma, an aggressive cancer with poor prognosis.

Normobaric hyperoxia combined with minocycline provides greater neuroprotection than either alone in transient focal cerebral ischemia

Normobaric hyperoxia (NBO), which maintains penumbral oxygenation, reduces brain injury during cerebral ischemia, and minocycline, a tetracycline derivative, reduces reperfusion injury, including inflammation, apoptosis and matrix metalloproteinases (MMPs) activation. Since they have different mechanisms of action, we hypothesized that combining them would provide greater neuroprotection. To test the hypothesis, we evaluated the neuroprotective effects of the combination of NBO with minocycline. Male Sprague–Dawley rats were exposed to NBO (95% O2) or normoxia (21% O2) during 90-min filament occlusion of the middle cerebral artery, followed by 48h of reperfusion. Minocycline (3mg/kg) or vehicle was intravenously administered to rats 15min after reperfusion onset. Treatment with NBO and minocycline alone resulted in 36% and 30% reductions in infarction volume, respectively. When the two treatments were combined, there was a 68% reduction in infarction volume. The combination therapy also significantly reduced hemispheric swelling, which was absent with monotherapy. In agreement with its greater neuro- and vasoprotection, the combination therapy showed greater inhibitory effects on MMP-2/9 induction, occludin degradation, caspase-3 and -9 activation and apoptosis inducing factor (AIF) induction in ischemic brain tissue. Our results show that NBO plus minocycline effectively reduces brain injury in transient focal cerebral ischemia with protection due to inhibition on MMP-2/9-mediated occludin degradation and attenuation of caspase-dependent and independent apoptotic pathways.

Type of serum influences the rituximab dependent cytotoxicity and apoptosis of chronic lymphocytic leukemia cells in vitro

The aim of the study was to compare the influence of types of serum on the in vitro viability and on either spontaneous or rituximab (RIT)-induced apoptosis of chronic lymphocytic leukemia (CLL) cells. The influence of fetal calf serum (FCS), patients' autologous serum (AS) and human AB-serum (ABS), used alone and in combinations consisting of two of them (v/v-1:1), on RIT-dependent cytotoxicity, apoptosis, detection of active forms of caspases-3,-9,-8 and disruption of mitochondrial membrane potential (ΔΨm) were assessed by flow cytometry. RIT was used at the concentration of 10 µg/ml. The spontaneous apoptosis was assessed in culture without RIT. AS revealed the protective action on CLL cells, however this serum added in vitro to the culture either alone or in combination with FCS was the only one to allow RIT to exert its cytotoxic action against CLL cells. RIT-induced apoptosis involved changes in ΔΨm and activation of caspases-3,-8,-9 when AS+FCS was applicated. Drug induced apoptosis (DIA) was 6.02 and 0.34, when FCS+AS and FCS alone were used, respectively (p<0.01). The RIT-dependent cytotoxic effect decreased when FCS+AS or FCS+ABS were used, as compared to effect of AS used separately. The cytotoxic effect of RIT did not depend on drug concentration, but on the type of serum added to the culture. The strongest cytotoxic effect of RIT in the presence of AS suggests that this drug activity towards CLL cells is enhanced by known cytotoxic mechanisms, caspase-dependent apoptotic pathway and possible influence of other extracellular factors present in the patients' sera.

Targeting a Novel Onco-glycoprotein Antigen at Tumoral Pancreatic Cell Surface by mAb16D10 Induces Cell Death

The mAb16D10 was raised against a pathological onco-glycoform of bile salt-dependent lipase isolated from the pancreatic juice of a patient suffering from a pancreatic adenocarcinoma. We previously showed that mAb16D10 specifically discriminates human pancreatic tumor tissues from other cancer and nontumor tissues. In this study, we report that mAb16D10 inhibited the proliferation of only human pancreatic tumor cells expressing 16D10 plasma membrane Ag. Interaction of mAb16D10 with its cognate surface Ag on pancreatic cells promoted cell death by activation of the p53- and caspase-dependent apoptotic pathway, and silencing of p53 decreased cell death. The decreased proliferation was also partly due to cell cycle arrest in G1/S phase, mAb16D10 triggering of glycogen synthase kinase-3β (GSK-3β) activation, degradation of β-catenin, and decreased expression of cyclin D1. GSK-3β positively affected p53 expression in pancreatic tumor cells after mAb16D10 binding. Inhibition of GSK-3β activity reversed the effects induced by mAb16D10 in SOJ-6 cells, supporting the pivotal role of GSK-3β signaling in the mechanisms of action induced by mAb16D10. Also, mAb16D10 cell treatment led to membrane overexpression of E-cadherin. Both E-cadherin and tumor Ag were localized in membrane lipid cholesterol-rich microdomains and are thought to belong to signaling platforms involved in the induction of cell cycle arrest and cell death. Overall, this study reveals that mAb16D10 holds great potential to prevent pancreatic tumor proliferation by apoptotic cell death, thus promising therapeutic prospects for treatment of pancreatic adenocarcinoma, a highly lethal disease.

Escherichia coli STb toxin induces apoptosis in intestinal epithelial cell lines

A previous study conducted in our laboratory demonstrated that cells having internalized Escherichia coli STb toxin display apoptotic-like morphology. We therefore investigated if STb could induce programmed cell death in both a human and an animal intestinal epithelial cell lines. HRT-18 (Human Colon Tumor) and IEC-18 (Rat Ileum Epithelial Cells) cell lines were used. As STb is frequently tested in a rat model, the IEC-18 cell line was most relevant to our work. The cell lines were treated with various amounts of purified STb (nanomole range) for a period of 24 h after which cells were harvested and examined for apoptotic characteristics. Caspase-9, the initiator of mitochondrion-mediated apoptosis, and caspase-3, an effector of caspase-9, were both activated following STb intoxication of HRT-18 and IEC-18 cells whereas caspase-8, the initiator caspase of the extrinsic pathway, was not activated. For both cell lines, agarose gel electrophoresis of the cell DNA content reveals laddering of DNA, resulting from DNA fragmentation, a characteristic of apoptosis. Hoechst 33342-stained DNA of STb-treated cell lines, observed using fluorescence microscopy, revealed condensation and fragmentation of the nuclei. Apoptotic indexes calculated from fragmented nuclei of Hoechst 33342-stained DNA for HRT-18 and IEC-18 cells showed an STb dose-dependent response. Overall, these data indicate that STb toxin induces a mitochondrion-mediated caspase-dependent apoptotic pathway.

인체 대장암 세포주 SW480에서 재배 와송의 세포 사멸 유도 효과

재배 와송의 항암효과를 입증하기 위해 SW480 대장암세포에서 재배 와송 추출물의 apoptosis 유도 효과 및 그 기전에 미치는 영향에 대해 알아보았다. 재배 와송과 천연 와송 메탄올 추출물의 암세포 성장 억제능을 측정한 결과, 재배 와송은 천연 와송과 유사하게 농도 의존적으로 높은 암세포 증식억제효과를 보였으며 특히 농도 300 <TEX>${\mu}g$</TEX>/mL의 재배 와송 메탄올 추출물에서는 천연 와송 메탄올 추출물 보다 더 높은 억제 효과를 나타내었다. 또한 재배 와송 메탄올 추출물을 분획하여 얻은 각각의 물, 헥산, 클로로포름, 에틸아세테이트 및 부탄올 분획물 중 와송 클로로포름 분획물에서 암세포의 증식 억제효과가 가장 높게 나타났다. 이러한 와송 클로로포름 분획물의 apoptosis 유도 효과는 Sub-G1기의 함량 증가와 핵의 응축 및 apoptotic bodies 생성 그리고 DNA 분절을 통해 나타났다. 한편, 와송 클로로포름 분획물의 caspase 활성은 caspase inhibitor 처리군에서 암세포의 증식 억제효과가 확연히 저해됨을 나타내었으며 각각의 caspase 활성 중 caspase-3 및 -9의 활성을 증가시켰다. 또한 와송 클로로포름 분획물은 apoptosis 관련 단백질들인 Bid, Bcl-2 및 PARP의 발현을 농도 의존적으로 감소시켰으며 tBid 및 Bax 단백질의 발현을 현저하게 증가시켰다. 본 연구 결과, 재배 와송과 천연 와송은 유사한 암세포 성장 억제 효과를 보였으며 재배 와송의 클로로포름 추출물은 caspase 의존적인 경로를 통해 apoptosis를 일으켜 세포 사멸 효과를 나타내는 것을 확인할 수 있었다. This study was performed to elucidate the anticancer activities and the mechanism of chloroform fractions from cultivated Orostachys japonicus (CFCOJ) in human colon cancer cells. CFCOJ markedly decreased viable cell numbers in both a dose-dependent and time-dependent manner within SW480 cells. Cell death induced by CFCOJ increased cell populations in the sub-G1 phase, as well as the formation of apoptotic bodies, nuclear condensation, and induced DNA fragmentation. CFCOJ-induced apoptosis was associated with the activation of initiator caspase-8 and -9, as well as the effector caspase-3. CFCOJ also stimulated Bid cleavage, indicating that the apoptotic action of caspase-8-mediated Bid cleavage leads to the activation of caspase-9. CFCOJ increased the expression of the proapoptotic protein, Bax, and decreased the expression of the antiapoptotic protein, Bcl-2. These results indicate that CFCOJ exert anticancer effects on human colon cancer SW480 cells through a caspase-dependent apoptotic pathway.

Synthesis of kojic acid-derived copper-chelating apoptosis inducing agents

Three classes of kojic acid derivatives were synthesized and examined for their antiproliferative activity against HeLa cells. Both 8b and 11 co-treated with copper ion exhibited synergistic effect on the HeLa cell growth inhibition with GI50 values of 11.9 and 7.1 μM, respectively. Flow cytometric analysis of HeLa cells revealed that 11-Cu co-treatment induced the sub-G1 arrest in a dose-dependent manner, suggesting that the growth-inhibitory effect is attributed to DNA fragmentation. Moreover, western blot of HeLa cells cytosolic extracts displayed the cleavage of the 116-kDa protein poly(ADP-ribose) polymerase and activation of caspase-3 by the reduced level of the 32-kDa proenzyme, indicating that the caspase-dependent apoptotic pathway was involved. We further demonstrated that MAPK pathway regulators such as ERK and p38 were activated in response to 11-Cu co-treatment, suggesting that the intracellular oxidative stress was dramatically stimulated by the copper ion. Taken together, we have successfully synthesized kojic acid-derived copper-induced apoptotic agents.

Anti-CHMP5 single chain variable fragment antibody retrovirus infection induces programmed cell death of AML leukemic cells in vitro

Over-expressed CHMP5 was found to act as oncogene that probably participated in leukemogenesis. In this study, we constructed the CHMP5 single chain variable fragment antibody (CHMP5-scFv) retrovirus and studied the changes of programmed cell death (PCD) of AML leukemic cells after infection by the retrovirus. The anti-CHMP5 KC14 hybridoma cell line was constructed to generate monoclonal antibody of CHMP5. The protein expression of CHMP5 was studied using immunofluorescence analysis. pMIG-CHMP5 scFv antibody expressible retroviral vector was constructed to prepare CHMP5-scFv retrovirus. AML leukemic U937 cells were infected with the retrovirus, and programmed cell death was studied using confocal microscope, FCM and Western blot. We obtained a monoclonal antibody of CHMP5, and found the expression of CHMP5 was up-regulated in the leukemic cells. After U937 cells were infected with CHMP5-scFv retrovirus, CHMP5 protein was neutralized. Moreover, the infection resulted in a significant increase in apoptosis and necrosis of U937 cells. In U937 cells infected with CHMP5-scFv retrovirus, apoptosis-inducing factor (AIF)-mediated caspase-independent necrotic PCD was activated, but autophagic programmed cell death was not observed. Neither the intrinsic nor extrinsic apoptotic PCD pathway was activated. The granzyme B/perforin-mediated caspase-dependent apoptotic PCD pathway was not activated. CHMP5-scFv retrovirus can neutralize the abnormally high levels of the CHMP5 protein in the cytosol of AML leukemic U937 cells, thereby inducing the programmed cell death of the leukemic cells via AIF-mediated caspase-independent necrosis and apoptosis.

Cytotoxicity of quinone drugs on highly proliferative human leukemia T cells: Reactive oxygen species generation and inactive shortened SOD1 isoform implications

Drugs containing the quinone group were tested on hyperproliferative leukemia T cells (HLTC: Jhp and Jws) and parental Jurkat cells. Doxorubicin, menadione and adaphostin produced different effects on these cell lines. Rapid doxorubicin-induced cell death in Jurkat cells was mediated by caspase activation. Doxorubicin-induced cell death of HLTCs was delayed due to the absence of caspase-3 and -8 expression. Delayed HLTC cell death was mediated and triggered by the generation of reactive oxygen species (ROS). Other drugs containing quinone groups, such as menadione and adaphostin, were also tested on HLTC and both were toxic by a caspase-independent mechanism. The toxicity of these drugs correlated with the generation of the superoxide anion, which increased and was more effective in HLTCs than in parental Jurkat cells. Accordingly, SOD1 activity was much lower in HLTCs than in Jurkat cells. This lower SOD1 activity in HLTCs was associated not only with the absence of the wild-type (16kDa) SOD1 monomer but also with the presence of a shortened (14kDa) SOD1 monomer isoform. Moreover, the cytotoxicity of drugs containing the quinone group was prevented by incubation with manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a cell-permeable superoxide dismutase mimetic and a potent inhibitor of oxidation. These findings could explain the sensitivity of HLTCs to drugs containing the quinone group using a mechanism dependent on oxidative stress. These observations can also be useful to target hyperproliferative leukemias that are resistant to the classical caspase-dependent apoptotic pathway.

Abstract 4967: Altholactone induces caspase dependent and independent apoptosis of human colorectal cancer cells

Abstract Resistance of colorectal cancer (CRC) to the available chemotherapy reveals the demand for identification of new anticancer agents. We evaluated the antitumor potential of altholactone, a naturally occurring bioactive compound isolated from Goniothalamus spp. (Annonaceae) hooks, against CRC cells. Antitumor activity of altholactone was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the propidium iodide method. Apoptosis mediators involved were assessed using biochemical inhibitors and Western blotting analysis. Results revealed that altholactone induced varying degrees of apoptosis in CRC cells but not in normal fibroblasts. Dissection of the altholactone-induced apoptotic signalling pathway revealed that altholactone activated caspase-dependent and -independent apoptotic pathways. Activation of caspase-4 appeared to be the initiating event in the caspase-dependent apoptotic pathway. Pre-treatment of CRC cells with the antioxidant N-acetylcysteine (NAC) significantly inhibited activation of caspase-4 and altholactone-induced apoptosis. These results indicate that altholactone induces selective cytotoxicity against colon carcinoma cells and warrants further clinical evaluation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4967. doi:1538-7445.AM2012-4967

Xanthohumol Induces Different Cytotoxicity and Apoptotic Pathways in Malignant and Normal Astrocytes

Cytotoxicity and the mechanisms of cell death induced by xanthohumol (XN) were compared in normal and cancerous human cells as the differences may be relevant for the potential use of XN in cancer therapy. The cancer cells seemed to be more susceptible to the cytotoxicity of XN than normal cells, but a significant difference was observed only in astrocytic cells. XN induced a higher rate of apoptosis in glioblastoma cells than in normal astrocytes, which was associated with activation of p53 and an elevated Bax/Bcl-2 ratio in glioblastoma cells, indicating an intrinsic caspase-dependent apoptotic pathway. In contrast, a reduced Bax/Bcl-2 ratio was observed in normal human astrocytes. This was also associated with higher expression of the cell cycle inhibitor, p21, in glioblastoma cells than in normal astrocytes. In addition, at a lower, non-cytotoxic concentration, XN partially inhibited the invasiveness of glioblastoma cells. Due to the selective sensitivity of astrocytic cells to XN, this compound should be studied further as a candidate for adjuvant therapy in the treatment of glioma.

Antitumor effects of ginsenoside Rg3 on human hepatocellular carcinoma cells

The antitumor effects of ginsenoside Rg3 have been reported in several kinds of human malignant tumors. The purpose of this study was to investigate whether ginsenoside Rg3 can inhibit the growth of human hepatocellular carcinoma cell lines and to discuss the possible molecular mechanism(s). We cultured the human hepatocellular carcinoma cell lines, SMMC-7721 and HepG2. The cells were treated with different concentrations of ginsenoside Rg3 (0, 25, 50, 75 and 100 µg/ml), and the cell proliferation was detected by MTT assay at the 12, 24, 36 and 48 h time-points. Flow cytometry experiments were carried out to investigate the effect of Rg3 on cell apoptosis after the cells had been treated with Rg3 (50 and 100 µg/ml) for 24 and 48 h. The expression levels of caspase-3, bax and bcl-2 in Rg3-treated cells (100 µg/ml, 48 h), as well as normal cells were detected through real-time PCR experiments. MTT assay showed that the inhibition rate of cell proliferation in the Rg3 groups was significantly higher compared to the control groups in both the SMMC-7721 and HepG2 cell lines, and the inhibition rate increased with increasing Rg3 concentrations and duration of treatment. Flow cytometry analysis demonstrated that the Rg3 groups had a significantly higher cell apoptotic rate compared to the control groups in both the SMMC-7721 and HepG2 cell lines, and that the effect of Rg3 on cell apoptosis occurred in a concentration- and time-dependent manner, as was also shown by the MTT assay. Real-time PCR analysis showed that the gene expression levels of caspase-3 and bax were significantly enhanced in the Rg3 groups compared to the control groups in both the SMMC-7721 and HepG2 cell lines, but the gene expression level of bcl-2 was significantly inhibited. These results indicate that ginsenoside Rg3 can effectively inhibit the growth of human hepatocellular carcinoma cell lines by inhibiting cancer cell proliferation and promoting cancer cell apoptosis, and it may promote cancer cell apoptosis via the endogenous mitochondrial-mediated caspase-dependent apoptotic pathway.

Simultaneous modulation of COX‐2, p300, Akt, and Apaf‐1 signaling by melatonin to inhibit proliferation and induce apoptosis in breast cancer cells

Melatonin exhibits anti-inflammatory and anticancer effects and could be a chemopreventive and chemotherapeutic agent against cancers, but the precise mechanisms involved remain largely unresolved. In this study, we evaluated the mechanism of action of melatonin in human MDA-MB-361 breast cancer cells. Melatonin at pharmacological concentrations (10(-3) m) significantly suppressed cell proliferation and induced apoptosis in a dose-dependent manner. The observed suppression of proliferation was accompanied by the melatonin-mediated inhibition of COX-2, p300, and NF-κB signaling. Melatonin significantly inhibited COX-2 expression and prostaglandin E(2) (PGE2) production, abrogated p300 histone acetyltransferase activity and p300-mediated NF-κB acetylation, thereby blocking NF-κB binding and p300 recruitment to COX-2 promoter. Pretreatment with a COX-2- or p300-selective inhibitor abrogated the melatonin-induced inhibition of cell proliferation, whereas PGE2 treatment or COX-2 transfection reversed the inhibition by melatonin. Moreover, melatonin markedly inhibited phosphorylation of PI3K, Akt, PRAS40, and GSK-3 proteins, thereby inactivating the PI3K/Akt signaling pathway. Pretreatment with a PI3K- or an Akt-selective inhibitor or an Akt-specific siRNA blocked the melatonin-mediated inhibition of cell proliferation. Conversely, gene delivery of a constitutively active Akt effectively reversed the inhibition by melatonin. Furthermore, melatonin induced Apaf-1 expression, triggered cytochrome C release, and stimulated caspase-3 and caspase-9 activities and cleavage, leading to an activation of the Apaf-1-dependent apoptotic pathway. Pretreatment with an Apaf-1-specific siRNA effectively attenuated the melatonin-induced apoptosis. These results therefore indicate that melatonin inhibits cell proliferation and induces apoptosis in MDA-MB-361 breast cancer cells in vitro by simultaneously suppressing the COX-2/PGE2, p300/NF-κB, and PI3K/Akt/signaling and activating the Apaf-1/caspase-dependent apoptotic pathway.