Levels Of Apoptosis Induction Research Articles

Discovery of selective CDK9 degraders with enhancing antiproliferative activity through PROTAC conversion.

Cyclin-dependent kinase 9 (CDK9) is an increasingly important potential cancer treatment target. Nowadays, developing selective CDK9 inhibitors has been extremely challenging as its ATP-binding sites are similar with other CDKs. Here, we report that the CDK9 inhibitor BAY-1143572 is converted into a series of proteolysis targeting chimeras (PROTACs) which leads to several compounds inducing the degradation of CDK9 in acute myeloid leukemia cells at a low nanomolar concentration. In addition, the most potent PROTAC molecule B03 could inhibit cell growth more effectively than warhead alone, with little inhibition of other kinases. This enhanced antiproliferative activity is mediated by a slight increase in kinase inhibitory activity and an increase in the level of apoptosis induction. Moreover, B03 could induce the degradation of CDK9 invivo. Our work provides evidence that B03 represents a lead for further development and that CDK9 degradation is a potential valuable therapeutic strategy in acute myeloid leukemia.

Fenretinide-polyethylene glycol (PEG) conjugate with improved solubility enhanced cytotoxicity to cancer cell and potent in vivo efficacy

Fenretinide (4-HPR), a synthetic retinoid, has shown its antitumor activity in many tumor types with low cytotoxicity to normal cells and high clinical safety. However, the low water solubility limits its further biological applications. To increase solubility, 4-HPR was conjugated with methoxy polyethylene glycol carboxylic acid (mPEG2K-COOH) by an ester linkage between the phenol hydroxyl of 4-HPR and the carboxyl of mPEG2K-COOH. The 4-HPR-PEG2K conjugate micelles had mean size of 76.70 ± 1.248 nm with a narrow distribution and a low critical micelle concentration. In vitro cytotoxicity studies showed the micelles have higher cytotoxicity to A2780s and MCF-7 cells. Its IC50 was 4.7 and 4.1-fold lower than the free 4-HPR, respectively. Importantly, in vivo pharmacokinetic studies, the AUC of 4-HPR was found to be 2.3-fold higher in 4-HPR-PEG2K micelles compared to free 4-HPR. And the 4-HPR-PEG2K micelles had higher antitumor activity. Meanwhile, the histopathology analysis exhibited that the micellar treatment decreased the viability of A2780s cells and increased the level of induced apoptosis. Therefore, the enhanced activity of 4-HPR by the method of conjugation with mPEG2K-COOH could hopefully provide new insights into the matter of ovarian cancer and breast cancer treatment.

The IN VITRO STUDY OF APOPTOSIS INDUCTION BY L-ASPARAGINASE OF ASPERGILLUS FUMIGATUS (ASPF-6) ON HUMAN LEUKEMIA CELL LINES

Objective: L-Asparaginase is a relatively widespread enzyme used in the lymphoblastic leukemia chemotherapy. In the present investigation, we report new potential fungal L-asparaginase producer Aspergillus fumigatus from Kadalundi mangrove forest, Kerala. The aim of the present investigation was to evaluate the concentration-dependent cytotoxicity and apoptosis-inducing the effect of extracted enzyme and its ability to induce apoptosis against human leukemia cell lines-HL-60.
 Methods: The fungal strain was isolated by serial dilution method from rhizosphere soil of mangrove forest. The isolated enzyme purified by 80% ammonium sulfate precipitation, dialysis, and ion exchange chromatography. To determine cytotoxicity and level of apoptosis, three different tests were performed: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, JC-1 flow cytometry, and Annexin V/propidium iodide (PI) flow cytometric DNA fragmentation. The test compound efficiency compared with positive drug doxorubicin against HL-60 cell lines.
 Results: The test compound exhibited a concentration-dependent cytotoxic effect on proliferation of HL-60 cell lines (IC-50 12.39 U) with a different level of apoptosis induction (LR-11.3%).
 Conclusion: A. fumigatus derived L-asparaginase may be clinically useful and results in better utilization for limited fungal-derived drug resources and improve financial feasibility as a therapeutic option for human leukemia.

Combinatorial effects of radiofrequency hyperthermia and radiotherapy in the presence of magneto‐plasmonic nanoparticles on MCF‐7 breast cancer cells

Here, the effects of combinatorial cancer therapy including radiotherapy (RT) and radiofrequency (RF) hyperthermia in the presence of gold-coated iron oxide nanoparticles (Au@IONPs), as a thermo-radio-sensitizer, are reported. The level of cell death and the ratio of Bax/Bcl2 genes, involved in the pathway of apoptosis, were measured to evaluate the synergistic effect of Au@IONPs-mediated RF hyperthermia and RT. MCF-7 human breast adenocarcinoma cells were treated with different concentrations of Au@IONPs. After incubation with NPs, the cells were exposed to RF waves (13.56 MHz; 100 W; 15 min). At the same time, thermometry was performed with an infrared (IR) camera. Then, the cells were exposed to 6 MV X-ray at various doses of 2 and 4 Gy. MTT (3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide) assay was performed to evaluate cell viability and quantitative real-time polymerase chain reaction (qRT-PCR) was used to determine the expression ratio of Bax/Bcl2. Cellular uptake of nanoparticles was confirmed qualitatively and quantitatively. The results obtained from MTT assay and qRT-PCR studies showed that NPs and RF hyperthermia had no significant effect when applied separately, while their combination had synergistic effects on cell viability percentage and the level of apoptosis induction. A synergistic effect was also observed when the cancer cells were treated with a combination of NPs, RF hyperthermia, and RT. On the basis of the obtained results, it may be concluded that the use of magneto-plasmonic NPs in the process of hyperthermia and RT of cancer holds a great promise to develop a new combinatorial cancer therapy strategy.

Synthesis and Biological Evaluation of New 1,3,4-Oxadiazoles as Potential Anticancer Agents and Enzyme Inhibitors.

1,3,4-Oxadiazoles have been known with a wide variety of pharmacological activities including anticancer activity. In this study, novel 2,5-disubstituted 1,3,4-oxadiazole derivatives were synthesized and evaluated for determining their anticancer, anticholinesterase and lipoxygenase (LOX) inhibitory activity. All compounds were tested against C6 rat glioma, A549 human lung carcinoma and NIH/3T3 mouse embryo fibroblast cell lines to define cytotoxic concentrations and apoptosis induction levels which they cause. Many of the compounds exhibited remarkable potency that compounds 2a, 2b, 2e, 2h and 2j against C6 cells and compounds 2a, 2b, 2d, 2g, 2i, 2j against A549 cells were found more active than cisplatin. Compound 2d namely, 2-[(5-(4-aminophenyl)-1,3,4-oxadiazol-2-yl)thio]-1-(4-chlorophenyl)ethan-1-one induced apoptosis of A549 cells with 74.9% whereas cisplatin caused 70.9% percentage. Among them, compounds 2d and 2j against A549 cell line, compounds 2b and 2e against both tumor cell lines showed selective cytotoxicity evaluating the inhibition concentration against NIH/3T3 cell line. None of the compounds showed significant acetylcholinesterase (AChE) and lipoxygenase inhibitory activities.

Selective heat generation in cancer cells using a combination of 808 nm laser irradiation and the folate-conjugated Fe2O3@Au nanocomplex

Photothermal therapy (PTT) is a nanotechnology-assisted cancer hyperthermia approach in which the interaction between laser light and plasmonic nanoparticles (NPs) generates localized heating. The exploitation of plasmonic NPs in association with active targeting moieties causes the preferential accumulation of NPs inside cancer cells, thereby providing targeted PTT. Herein, we evaluate the effect of folic acid (FA) as an active targeting agent in enhancing the photothermal efficiency of multifunctional Iron (III) Oxide (Fe2O3)@Au core- shell NPs. Fe2O3@Au NPs were synthesized, modified with FA and then characterized. Human nasopharyngeal (KB) cancer cells were treated with different concentrations of Fe2O3@Au, with and without FA modification and the temperature rise profiles of the cells were measured upon administration of the near-infrared (NIR) laser (808 nm, 6 W/cm2, 10 min). The recorded temperature profiles of the cells were used for thermal dose calculation. Finally, the level of induced apoptosis was determined by flow cytometry using an annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit. The characterization data showed that the Fe2O3@Au NPs are spherical, with a hydrodynamic size of 33 nm. The data corroborated the successful conjugation of the NPs with FA. The thermometry results indicated the superior temperature elevation rate of the cells in the presence of the NPs upon NIR irradiation. Meanwhile, the higher heating rate and the higher thermal dose were obtained for the cells exposed to FA-targeted Fe2O3@Au rather than the non-targeted nanocomplex. Flow cytometry studies revealed that FA-targeted Fe2O3@Au induced higher level of apoptosis than non-targeted Fe2O3@Au NPs. In conclusion, our findings suggest that the synthesized FA-targeted Fe2O3@Au NP has high potentials to be considered as an efficient thermosensitizer in the process of targeted cancer hyperthermia.

Reactive oxygen species-dependent apoptosis induction by water extract of Citrus unshiu peel in MDA-MB-231 human breast carcinoma cells.

BACKGROUND/OBJECTIVESAlthough several recent studies have reported the anti-cancer effects of extracts or components of Citrus unshiu peel, which has been used for various purposes in traditional medicine, the molecular mechanisms for their effects remain unclear. In the present study, the anti-cancer activity of a water-soluble extract of C. unshiu peel (WECU) in MDA-MB-231 human breast carcinoma cells at the level of apoptosis induction was investigated.MATERIALS/METHODSCytotoxicity was evaluated using the MTT assay. Apoptosis was detected using DAPI staining and flow cytometry analyses. Mitochondrial membrane potential, reactive oxygen species (ROS) assay, caspase activity and Western blotting were used to confirm the basis of apoptosis.RESULTSThe results indicated that WECU-induced apoptosis was related to the activation of caspase-8, and -9, representative initiator caspases of extrinsic and intrinsic apoptosis pathways, respectively, and caspase-3 accompanied by proteolytic degradation of poly(ADP-ribose) polymerase and down-regulation of the inhibitors of apoptosis protein family members. WECU also increased the pro-apoptotic BAX to anti-apoptotic BCL-2 ratio, loss of mitochondrial membrane potential and cytochrome c release from mitochondria to cytoplasm. Furthermore, WECU provoked the generation of ROS, but the reduction of cell viability and induction of apoptosis by WECU were prevented when ROS production was blocked by antioxidant N-acetyl cysteine.CONCLUSIONSThese results suggest that WECU suppressed proliferation of MDA-MB-231 cells by activating extrinsic and intrinsic apoptosis pathways in a ROS-dependent manner.

An active molecule from Pulsatilla chinensis, Pulsatilla saponin A, induces apoptosis and inhibits tumor growth of human colon cancer cells without or with 5-FU.

Colon cancer is one of the common types of digestive malignancy. The efficacy of the first-line chemotherapy drug for colon cancer, fluorouracil (5-FU), remains limited in clinical settings due to poor efficacy and significant side effects. In the present study, the anticancer activity of an active compound from Pulsatilla chinensis extracts, Pulsatilla saponin A (PsA), was isolated and examined in vitro and in vivo. It was demonstrated that PsA significantly inhibited the growth of human colon cancer HT-29 cells. This inhibitory activity was also observed when the compound was tested in a colon cancer xenograft mouse model. Additionally, the synergic antitumor effects of PsA and 5-FU on colon cancer cells were observed. Using annexin V and terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling assays, it was demonstrated that levels of apoptosis induction in HT-29 cells treated with PsA or 5-FU were significantly increased compared with the untreated control cells (P<0.05). Western blot analyses were then performed, and the results revealed an increase in tumor protein 53 and cleaved caspase 9, and a decrease in B-cell lymphoma 2 protein expressions in PsA and PsA + 5-FU treated colon cancer cells compared with the vehicle-treated (PBS) cells. In summary, PsA exhibited anticancer activity in human colon cancer cells in vitro and in vivo, in isolation and synergistically with 5-FU, through apoptosis induction.

Pegylated Interferon-α2a Inhibits Proliferation of Human Liver Cancer Cells In Vitro and In Vivo

PurposeWe investigated the effects of pegylated interferon-α2a (PEG-IFN-α2a) on the growth of human liver cancer cells.MethodsThe effect of PEG-IFN-α2a on the proliferation of 13 liver cancer cell lines was investigated in vitro. Cells were cultured with medium containing 0–4,194 ng/mL of PEG-IFN-α2a, and after 1, 2, 3, or 4 days of culture, morphologic observation and growth assay were performed. After hepatocellular carcinoma (HCC) cells (HAK-1B and KIM-1) were transplanted into nude mice, various doses of PEG-IFN-α2a were subcutaneously administered to the mice once a week for 2 weeks, and tumor volume, weight, and histology were examined.ResultsPEG-IFN-α2a inhibited the growth of 8 and 11 cell lines in a time- and dose-dependent manner, respectively, although the 50% growth inhibitory concentrations of 7 measurable cell lines on Day 4 were relatively high and ranged from 253 ng/mL to 4,431 ng/mL. Various levels of apoptosis induction were confirmed in 8 cell lines. PEG-IFN-α2a induced a dose-dependent decrease in tumor volume and weight, and a significant increase of apoptotic cells in the tumor. Subcutaneous administration of clinical dose for chronic hepatitis C (3 μg/kg, 0.06 μg/mouse) was effective and induced about 30-50% reduction in the tumor volume and weight as compared with the control.ConclusionsAlthough in vitro anti-proliferative effects of PEG-IFN-α2a were relatively weak, PEG-IFN-α2a induced strong anti-tumor effects on HCC cells in vivo. The data suggest potential clinical application of PEG-IFN-α2a for the prevention and treatment of HCC.

Life history traits and functional immune parameters in the wild trout of the Kerguelen Islands

Common trouts (Salmo trutta L.) over 3 years old, were sampled in hydrosystems of Kerguelen Islands in 2010. Biometric and biogeographical data of caught fish were collected in the field. Ages and migratory status of individuals were determined by scale reading and validated by otolithometry and otolith microchemistry (LA-ICPMS). Prey types ingested by trout were determined by analysis of stomach contents. Molecules organochlorine pesticides (OCPs) and PCBs accumulated in the liver and muscle tissues of fish were analyzed by GC-ECD. The immunomarkers were measured directly in the field by appropriate protocols in flow cytometry. Five groups of trouts associated with distinct ecological niches, were defined by the nature of ingested preys. Groups 1 and 3 fed from only benthic invertebrates, group 2 from terrestrial origin preys (insects or arthropods drained by runoff waters), group 4 from pelagic invertebrates (insect larvae) and the group 5 fed exclusively from marine organisms. High significant variations in oxidative burst of blood and pronephros leukocytes were observed between groups. It was particularly true for trouts whose diet consisted in benthic or terrestrial preys which had the highest levels in leukocyte oxidative burst. In addition, a negative influence of hepatic contamination by DDT, HCB and PCB was observed on oxidative burst of pronephrotic leukocytes. The basal level of apoptosis in pronephrotic leukocytes was strongly correlated with muscle bioaccumulation of OCPs, PCBs and PBDEs. Levels of induced apoptosis were particularly high in the blood or in the pronephros of trouts predating preferentially pelagic organisms. This factor was positively correlated with the levels of hepatic OCP, such as DDT. Chronic contamination of trout was also associated with low blood phagocytic activity in migratory trout feeding exclusively marine organisms. In the natural environment, the ecological niche occupied by the fish influence their chemical contamination and plays a major role in the expression levels of immune cell functionality.

Microarray determination of Bcl-2 family protein inhibition sensitivity in breast cancer cells

This study tests the hypothesis that reverse transcription polymerase chain reaction (RT-PCR) microarrays can be used to predict the relative sensitivity to induction of apoptosis in breast cancer cells exposed to inhibitors of antiapoptotic Bcl-2 family proteins. Four cell lines, MDA-MB-231 (MDA-231) and MDA-MB-468 (MDA-468), BT-20 and T47-D were screened for relative expression of Bcl-2 family members A1, Mcl-1, Bcl-2, Bcl-xL and Bcl-w mRNA by RT-PCR microarrays and Western analysis. The four cell lines were treated with 1 μmol/L obatoclax (GX15-070) and/or 2 Gy radiation (RT) and monitored for apoptosis after 48 h. Cell lines showing the highest total fold-increase of Bcl-2 family member mRNA, MDA-231 and MDA-468, also showed the highest levels of apoptosis induction (approximately 70% with obatoclax alone and 82% with obatoclax plus RT). Cell lines with little or no increase in Bcl-2 family mRNA (BT-20 and T47-D) showed less apoptosis (30% following treatment with obatoclax and 42% with obatoclax plus RT). RT-PCR arrays can predict the relative apoptosis response of breast cancer cells to the pan Bcl-2 inhibitor obatoclax alone or when combined with radiation.

Эффективность фракционированного воздействия γ-излучения и быстрых нейтронов на саркому М-1

The effectiveness of fractionated exposure to gamma- and neutron radiation in their separate and combined use on the growth and functional morphology of mutant p53 sarcoma M-1 in rats was studied. Investigation techniques included immunostaining of PCNA and mutant p53 expressing cells, determination of mitotic activity and apoptotic death of tumor cells, as well as computer analysis of microscopic images. The antitumor efficacy of different types of radiation is shown to be determined by different levels of apoptosis induction, reduced proliferation and cellularity. Neutron radiation of the impulse generator has a marked damaging effect on the vasculature and the development of tumor necrosis. Fractionated irradiation at equal daily doses led to the decrease in the relative effectiveness of radio-inactivation of tumor cells. After 9 fractions of irradiation, the calculated value of the RBE of fast neutrons normalized to the input dose of 1 Gy by the coefficient of tumor growth inhibition, a reduced proliferative activity of PCNA and induced apoptosis of tumor cells was 3.4, 3.7 and 3.1, respectively. In the mode of daily superfractionation with splitting the dose in two fractions, the effectiveness of the combined exposure corresponded to the additive effect of gamma- and neutron radiation with a tendency toward synergism. There are reasons to believe that high resistance of sarcoma M-1 to the ionizing radiation impact is due not only to a fraction of hypoxic cells, but also the mutant status of p53 gene.

Specific features of the apoptotic response of urinary bladder cancer cells to neoadjuvant chemotherapy

The TUNEL reaction was used to study induced apoptosis in the tumor cells of urinary bladder cancer (UBC) patients. It has been shown that an increase in the values of induced apoptosis correlates well with the indicators showing a tumor’s corresponding response to the application of neoadjuvant chemotherapy, using the gemcitabine-cisplatin regimen. It has been concluded that assessing the level of induced apoptosis in UBC tumor cells using the TUNEL assay enables making a prognosis for the efficacy of chemotherapy at the single-cell level in patients with this type of cancer.

The cisplatin–irradiation combination suggests that apoptosis is not a major determinant of clonogenic death

It is commonly believed that tumor cells treated with anticancer agents, chemotherapy and/or radiation, die by apoptosis and that tumors which do not undergo apoptosis are resistant to treatment. In this study, we investigated the molecular basis underlying cisplatin cytotoxicity in the murine teratocarcinoma F9 cell line to see whether irradiation enhances cisplatin-induced cytotoxicity. We compared the apoptosis induced by chemo and/or radiotherapy with other cellular effects such as cell survival, clonogenic capability, cell cycle perturbation, expression of p53 and p53-related mRNAs, and necrosis. When combined with radiation, a clear additive cytotoxic effect of cisplatin was demonstrated. We found that both cisplatin and radiation induced cell death, but the level of induced apoptosis was low and there was no correlation with the results of the clonogenic assays: we noted a difference between cytotoxic effects in the clonogenic assay and the extent of apoptosis by fluorescence-activated cell sorter analysis, suggesting that cell killing reflected not only apoptosis but also cell cycle arrest, and that apoptosis, cell kinetics and clonogenicity suppression were independent processes.

Bcl-2 over-expression promotes genomic instability by inhibiting apoptosis of cells exposed to hydrogen peroxide

The anti-apoptotic oncogene bcl-2 is hypothesized to increase the antioxidant status of cells, thereby protecting them from oxidative stress. In this study, we examined hydrogen peroxide (H2O2)-mediated oxidative stress in Jurkat T lymphoma cells. Over-expression of Bcl-2 did not inhibit cytotoxicity at doses of H2O2 that caused necrosis (>200 microM), but it did block cell death at apoptotic doses (<200 microM). However, these cells exhibited the same initial level of protein and lipid oxidation following exposure to H2O2 as the parental cells, indicating that the anti-apoptotic activity is not associated with general antioxidant properties. Bcl-2 expression was able to protect against secondary protein carbonyl formation, which was linked to lysosome stabilization. Assessment of micronuclei formation in cells over-expressing Bcl-2 showed evidence of increased genomic instability, consistent with the impairment of apoptosis in damaged cells. We conclude that while Bcl-2 can block cytotoxicity associated with apoptosis-inducing levels of oxidative stress, it does not protect the cells from the stress itself. Bcl-2 may promote tumourigenesis by preventing the removal of oxidatively damaged cells.

The Role of Vpr in HIV-1 Disease Progression is Independent of Its G2 Arrest Induction Function

Vpr (viral protein R) is a vital HIV-1 accessory protein with multiple functions in the viral life cycle, including nuclear import of preintegration complex, induction of apoptosis and G2 cell cycle arrest. The cell cycle perturbation activity of Vpr requires activation of the ATR (Ataxia-Telangiectasia and Rad3-related) pathway and the integrity of Vpr C-terminal motif that is crucial for chromatin binding. Recent studies also demonstrated Vpr as one of the viral factors that influence HIV disease progression, as mutations in Vpr were overrepresented in some cohorts of long-term nonprogressors (LTNP). The LTNP-associated mutations of Vpr are frequently observed in the C-terminal domain. This raises the question whether the LTNP phenotype of Vpr is the result of the loss its ability to induce G2 arrest. Here we report that the LTNP-associated mutants of Vpr function normally in the induction of G2 arrest. No defects in ATR activation and direct binding to chromatin are observed. These mutants also show similar levels of apoptosis induction as wild-type Vpr. These data differentiate the LTNP-associated mutations of Vpr with those defective in inducing G2 arrest. We propose that the G2 arrest function of Vpr is separated from the LTNP phenotype, and the role of Vpr in HIV disease progression may involve other functions of Vpr.

Acidic fibroblast growth factor decreases α-smooth muscle actin expression and induces apoptosis in human normal lung fibroblasts

Fibroblast/myofibroblast expansion is critical in the pathogenesis of pulmonary fibrosis. To date, most research has focused on profibrotic mediators, whereas studies on antifibrotic factors are scanty. In this study, we explored the effects of acidic fibroblast growth factor (FGF-1) and FGF-1 plus heparin (FGF-1+H) on fibroblast growth rate, apoptosis, and myofibroblast differentiation. Heparin was used because it participates in FGF-1 signaling. Growth rate was evaluated by WST-1 colorimetric assay, DNA synthesis by [(3)H]thymidine incorporation, and apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and cleaved caspase 3. Expression of alpha-smooth muscle actin (alpha-SMA) was examined by immunocytochemistry, flow cytometry, real-time PCR, and immunoblotting. Despite the induction of DNA synthesis, FGF-1+H significantly reduced fibroblast growth rate. This correlated with a significant increase in apoptosis, evaluated by TUNEL (41.6 +/- 1.4% vs. 12.5 +/- 0.6% from controls; P < 0.01) and cleaved caspase 3 (295 +/- 32 vs. 200 +/- 19 ng/10(6) cells from controls; P < 0.05). Double immunostaining (alpha-SMA-TUNEL) revealed that the levels of induced apoptosis were similar in fibroblasts and myofibroblasts. FGF-1+H inhibited the effect of TGF-beta1 on myofibroblast differentiation. alpha-SMA-positive cells were reduced by immunocytochemistry from 44.5 +/- 6.5% to 10.9 +/- 1.9% and by flow cytometry from 30.6 +/- 2.5% to 7.7 +/- 0.6% (P < 0.01). Also, FGF-1+H significantly inhibited the TGF-beta1 induction of alpha-SMA quantified by real-time PCR and Western blot. This decrease was associated with a 35% reduction in TGF-beta1-induced collagen gel contraction. The effect of FGF-1+H was mediated by a significant decrease of TGF-beta1-induced Smad2 phosphorylation. FGF-1 alone exhibited similar but lower effects. These findings suggest that FGF-1 can have an antifibrogenic role, inducing apoptosis of fibroblasts and inhibiting myofibroblast differentiation.

Profiling Tumor Counterattack: Do Fas Ligand–Containing Microvesicles Reduce Anticancer Immunity?

Profiling Tumor Counterattack: Do Fas Ligand–Containing Microvesicles Reduce Anticancer Immunity?

FR901228 induces tumor regression associated with induction of Fas ligand and activation of Fas signaling in human osteosarcoma cells.

We investigated the antitumor effects of FR901228, a HDAC inhibitor, on human osteosarcoma cells, in vitro and in vivo to explore its possible utility in the treatment of pediatric bone cancers. FR901228 caused marked growth inhibition with a 50% inhibitory concentration of 1.2-7.3 nM and induction of apoptosis in all eight osteosarcoma cell lines tested. These effects of FR901228 were also observed in vivo xenograft models on BALB/c nude mice, and treatment with 5.6 mg/kg/day resulting in a >70% reduction in the mean final tumor volume compared with the mean initial tumor volume. TUNEL assays demonstrated extensive apoptosis in tumor sections of mice treated with FR901228. Induction of apoptosis was preceded by increased expression of Fas ligand (FasL) mRNA, resulting in expression of membrane-bound FasL, which was followed by sequential activation of caspase-8 and -3. The level of apoptosis induction was reduced using a neutralizing anti-FasL antibody and overexpression of either the dominant-negative FADD or the viral FLICE inhibitory protein. Furthermore, treatment with a suboptimal dose of FR901228 greatly sensitized osteosarcoma cells to agonistic anti-Fas antibody-mediated apoptosis. These findings suggest that FR901228 is a highly promising antitumor agent against osteosarcoma, inducing apoptosis by the activation of the Fas/FasL system.

The vpu protein of human immunodeficiency virus type 1 plays a protective role against virus-induced apoptosis in primary CD4(+) T lymphocytes.

Previous data revealed that primary cultures of peripheral blood mononuclear cells (PBMCs) were killed by apoptosis at higher rates after infection with two CRF01_AE primary isolates of human immunodeficiency virus type 1 (HIV-1) than after infection with five other CRF01_AE primary isolates, five subtype B primary isolates, and two subtype B laboratory strains. Here, we show evidence that mutations at the vpu gene which were exclusively identified only in the two CRF01_AE isolates mentioned above are involved in their abilities to induce massive apoptosis in primary CD4(+) T lymphocytes. The rates of virus production by these two isolates in the culture media of infected PBMCs were lower (the same as those of the other CRF01_AE isolates) than those of the subtype B isolates. To confirm the correlation between the higher apoptosis-inducing abilities and the mutations at the vpu gene, infectious molecular clone pNL4-3-based vpu mutants were constructed and examined for their apoptosis induction levels. The apoptosis induction levels after introduction of the vpu mutations were greatly increased in primary CD4(+) T lymphocytes. In contrast, the apoptosis induction abilities of these vpu mutants were lower in human T-cell line MT-4. Thus, the Vpu protein of HIV-1 could play a protective role against virus-induced apoptosis in primary CD4(+) T lymphocytes.