Curcumin differentially sensitizes malignant glioma cells to TRAIL/Apo2L-mediated apoptosis through activation of procaspases and release of cytochrome c from mitochondria

Malignant glioma cells are generally resistant or only weakly sensitive to tumor necrosis factor family of cell death-inducing ligands, including TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L. The chemopreventive activity of polyphenolic compounds present in plant-derived food products has been well recognized in epidemiological studies; however, the mechanism of chemoprevention by these dietary constituents largely remains unknown. Curcumin, the yellow pigment in the spice turmeric, has profound anti-inflammatory activity and exhibits chemopreventive and tumor growth inhibitory activity. In the present study, we investigated whether curcumin sensitizes malignant glioma cell lines U251MG and U87MG to TRAIL-induced apoptosis. Treatment with low concentrations (5-20 microM) of curcumin alone had no effect on the viability of either cell line. At low concentration (5 ng/ml) TRAIL induced cytotoxicity in U251MG cells but not in U87MG cells. Whereas curcumin at subtoxic concentration sensitized U87MG cells to TRAIL-induced cytotoxicity, it had no effect on TRAIL-mediated cytotoxicity in U251MG cells. The combined curcumin and TRAIL treatment enhanced accumulation of hypo-diploid U87MG cells in sub G1 cell cycle phase and induced the cleavage of procaspases-3, -8, -9 and release of cytochrome c from mitochondria. These data indicate that curcumin differentially sensitizes glioma cells to TRAIL-induced apoptosis through the activation of both extrinsic (receptor-mediated) and intrinsic (chemical-induced) pathways of apoptosis. These results define a potential use of curcumin to sensitize glioma cells for TRAIL-mediated immunotherapy.     

Sensitization to CD95 ligand-induced apoptosis in human glioma cells by hyperthermia involves enhanced cytochrome c release

CD95L-induced apoptosis involves caspase activation and is facilitated when RNA and protein synthesis are inhibited. Here, we report that hyperthermia sensitizes malignant glioma cells to CD95L- and APO2L-induced apoptosis in the absence, but not in the presence, of inhibitors of RNA and protein synthesis. Hyperthermia does not alter CD95 expression at the cell surface and does not modulate the morphology of CD95-mediated cell death on electron microscopy. Bcl-2 gene transfer inhibits apoptosis and abrogates the sensitization mediated by hyperthermia. Hyperthermia does not overcome resistance to apoptosis conferred by the viral caspase inhibitor, crm-A, indicating the absolute requirement for the activation of crm-A-sensitive caspases, probably caspase 8, for apoptosis. CD95L-evoked DEVD-amc-cleaving caspase activity is enhanced by hyperthermia, suggesting that hyperthermia operates upstream of caspase processing to promote apoptosis. There is no uniformly enhanced processing of three caspase 3 substrates, poly-ADP ribose polymerase (PARP), protein kinase C (PKC) delta and DNA fragmentation factor (DFF) 45. Yet, hyperthermia promotes CD95L-evoked DNA fragmentation. Interestingly, hyperthermia enhances the CD95L-evoked release of cytochrome c in the absence, but not in the presence, of CHX. In contrast, the reduction of the mitochondrial membrane potential is enhanced by hyperthermia both in the absence and presence of CHX, and enhanced cytochrome c release is not associated with significantly enhanced caspase 9 processing. The potentiation of cytochrome c release at hyperthermic conditions in the absence of CHX is abrogated by Bcl-2. Thus, either hyperthermia or inhibition of protein synthesis by CHX potentiate cytotoxic cytokine-induced apoptosis. These pathways show no synergy, but rather redundance, indicating that CHX may function to promote apoptosis in response to cytotoxic cytokines by inhibiting the synthesis of specific proteins whose synthesis, function or degradation is temperature-sensitive.     

A mechanism of resistance to TRAIL/Apo2L-induced apoptosis of newly established glioma cell line and sensitisation to TRAIL by genotoxic agents

Most tumour cells are sensitive to TRAIL-induced apoptosis, but not normal cells; thus, cancer therapy using TRAIL is expected clinically. Several tumour cells are resistant to TRAIL-induced apoptosis, and various mechanisms of such resistance were reported in individual cases. In this study, we established a TRAIL-resistant glioma cell line, which completely lacked TRAIL receptors. In addition, this tumour cell line had wild-type p53 tumour-suppressive gene, suggesting new mechanisms for tumour cells to expand and escape from immune surveillance. The present study further explored the mechanisms that determine the sensitivity to TRAIL. We show that genotoxic agents such as cisplatin, doxorubicin and camptothecin, in addition to UV radiation, can induce TRAIL-R2 on the cell surface of TRAIL receptor-negative tumour cells. Newly synthesised TRAIL-R2 is functional, so apoptosis is effectively induced by TRAIL, but it is significantly inhibited by constitutive expression of dominant-negative p53. In addition, apoptosis induced by pretreatment of genotoxic agents and additional stimulation of TRAIL is efficiently inhibited by either antagonistic anti-TRAIL-R2 antibody or pan-caspase inhibitor z-VAD-FMK. Taken together, these findings suggest that resistance to TRAIL by lack of TRAIL receptors on glioma is restored by genotoxic agents, which support the new strategies for tumour killing by TRAIL-bearing cytotoxic cells in combination with genotoxic treatment.     

Transcriptional activation of TRADD mediates p53-independent radiation-induced apoptosis of glioma cells.

Survival of patients with Glioblastoma Multiforme (GM), a highly malignant brain tumor, remains poor despite concerted efforts to improve therapy. The median survival of patients with GM has remained approximately 1 year regardless of the therapeutic approach. Since radiation therapy is the most effective adjuvant therapy for GM and nearly half of GM tumors harbor p53 mutations, we sought to identify genes that mediate p53-independent apoptosis of GM cells in response to ionizing radiation. Using broad-scale gene expression analysis we found that following radiation treatment, TRADD expression was induced in a uniquely radiosensitive GM cell line but not in radioresistant GM cell lines. TRADD over-expression killed GM cells and activated NF-kappa B. We found that blocking the TRADD-mediated pathway using a dominant-negative mutant of FADD (FADD-DN) enhanced radiation resistance of GM cells, as reflected in both susceptibility to apoptosis and clonogenic survival following irradiation. Conversely, stable expression of exogenous TRADD enhanced radiation-induced apoptosis of GM cell lines, reflecting the biological significance of TRADD regulation in p53-independent apoptosis. These findings generate interest in utilizing TRADD in gene therapy for GM tumors, particularly in light of its dual function of directly inducing rapid apoptosis and sensitizing GM cells to standard anti-neoplastic therapy.     

Rapamycin-induced apoptosis is p53-independent in human prostate carcinoma PC-3 cells

We have investigated the mechanisms of rapamycin-induced growth inhibition and apoptosis in the PC-3 prostate carcinoma cell line. Rapamycin induced apoptosis as well as the expression of p21(waf1) mRNA and protein, independent of p53. Rapamycin treatment also resulted in: a decrease in cdk2 kinase activity; an increase in hypophosphorylated retinoblastoma protein (pRb); a dephosphorylation of p70 S6 kinase; and, growth-arrest in G(1)-phase of cell cycle. These data suggest that rapamycin-induced growth arrest and apoptosis occur through the p53-independent induction of p21(waf1). Since this induction occurred soon after rapamycin treatment, possibly, the early induction of p21(waf1) and G(1)-arrest are important components of the mechanism by which rapamycin induces apoptosis in PC-3 cells.     

Mitochondrial cytochrome c release is caspase-dependent and does not involve mitochondrial permeability transition in didemnin B-induced apoptosis

Permeability transition, and a subsequent drop in mitochondrial membrane potential (DeltaPsi(m)), have been suggested to be mechanisms by which cytochrome c is released from the mitochondria into the cytosol during apoptosis. Furthermore, a drop in DeltaPsi(m) has been suggested to be an obligate early step in the apoptotic pathway. Didemnin B, a branched cyclic peptolide described to have immunosuppressive, anti-tumour, and anti-viral properties, induces rapid apoptosis in a range of mammalian cell lines. Induction of apoptosis by didemnin B in cultured human pro-myeloid HL-60 cells is the fastest and most complete ever described with all cells being apoptotic after 3 h of treatment. By utilizing the system of didemnin B-induced apoptosis in HL-60 cells, and the potent inhibitors of mitochondrial permeability transition, cyclosporin A and bongkrekic acid, we show that permeability transition as determined by changes in DeltaPsi(m) and mitochondrial Ca2+ fluxing, is not a requirement for apoptosis or cytochrome c release. In this system, changes in mitochondrial membrane potential and cytochrome c release are shown to be dependent on caspase activation, and to occur concurrently with the release of caspase-9 from mitochondria, genomic DNA fragmentation and apoptotic body formation.     

Caffeine enhanced radiosensitivity of rat tumor cells with a mutant-type p53 by inducing apoptosis in a p53-independent manner

The radiosensitizing effects of caffeine on two rat yolk sac tumor cell lines with a different p53 status were investigated. A reduction of radiation-induced G(2) arrest was caused by caffeine at a concentration of 2 mM in both cell lines. The reduction of survival was observed in a combination of radiation and 2 mM caffeine only in a lower radiation dose range, but not in a higher dose range in NMT-1 with a wild type p53. Radiosensitization of caffeine was recognized even in a higher dose range for cells with a mutant-type p53. Apoptosis, which was not prominent after irradiation alone or caffeine treatment alone, was induced by irradiation in combination with caffeine in cells with a mutant-type p53 through a p53-independent pathway.     

Betulin induces mitochondrial cytochrome c release associated apoptosis in human cancer cells

We examined whether betulin, a naturally abundant compound, has anticancer functions in human cancer cells. The results showed that betulin significantly inhibited cell viability in cervix carcinoma HeLa cells, hepatoma HepG2 cells, lung adenocarcinoma A549 cells, and breast cancer MCF‐7 cells with IC₅₀ values ranging from 10 to 15 µg/mL. While betulin exhibited only moderate anticancer activity in other human cancer cells such as hepatoma SK‐HEP‐1 cells, prostate carcinoma PC‐3, and lung carcinoma NCI‐H460, with IC₅₀ values ranging from 20 to 60 µg/mL, it showed minor growth inhibition in human erythroleukemia K562 cells (IC₅₀ > 100 µg/mL). We further investigated the mechanism of anticancer activity by betulin, using HeLa cells as an experimental model. Betulin (10 µg/mL) induces apoptotic cell death, as evidenced by morphological characteristics such as membrane phosphatidylserine translocation, nuclear condensation/fragmentation, and apoptotic body formation. A kinetics analysis showed that the depolarization of mitochondrial membrane potential and the release of mitochondrial cytochrome c occurred as early as 30 min after treatment with betulin. Betulin, unlike its chemical derivative betulinic acid, did not directly trigger mitochondrial cytochrome c release in isolated mitochondria. Importantly, Bax and Bak were rapidly translocated to the mitochondria 30 min after betulin treatment. The sequential activation of caspase‐9 and caspase‐3/‐7 and the cleavage of poly(ADP‐ribose) polymerase (PARP) were observed behind those mitochondrial events. Furthermore, specific downregulation of either caspase‐9, Bax, or Bak by siRNA effectively reduced PARP cleavage and caspase‐3 activation. Taken together, the lines of evidence demonstrate that betulin triggers apoptosis of human cancer cells through the intrinsic apoptotic pathway. © 2010 Wiley‐Liss, Inc.     

Requirement of BAX for TRAIL/Apo2L-induced apoptosis of colorectal cancers: synergism with sulindac-mediated inhibition of Bcl-x(L)

The cornerstone of the systemic treatment of advanced colorectal cancer is 5-fluorouracil.However, 5-fluorouracil-induced apoptosis is dependent on p53, a tumor suppressor gene that is lost or inactivated in at least 85% of human colorectal cancers. Here we show that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L triggers caspase-8-mediated truncation of BID, mitochondrial activation of caspase-9, and apoptosis in both p53(+/+) or p53(-/-) isogenic HCT116 colorectal cancer cells. TRAIL/Apo2L also sensitizes both p53(+/+) or p53(-/-) colorectal cancer cells to ionizing radiation. In contrast, we find that TRAIL/Apo2L fails to activate caspase-9 or induce apoptosis in isogenic HCT116 colorectal cancer cells that are deficient in BAX, a proapoptotic gene that is mutated in >50% of colorectal cancers of the microsatellite mutator phenotype. Loss of BAX also renders colorectal cancer cells resistant to TRAIL/Apo2L-mediated radiosensitization. We additionally demonstrate that TRAIL/Apo2L-induced death of p53(+/+)- or p53(-/-)- BAX-proficient but not BAX-deficient colorectal cancer cells is augmented by reducing nuclear factor-kappaB-dependent expression of Bcl-x(L) with either a peptide that disrupts the inhibitor of kappaB kinase complex or the nonsteroidal anti-inflammatory drug, sulindac sulfide. These results indicate that the combination of TRAIL/Apo2L with either irradiation or sulindac may be highly effective against both p53-proficient and p53-deficient colorectal cancers; however, BAX-deficient tumors may evade elimination by TRAIL/Apo2L-based regimens. Our findings may aid the development and genotype-specific application of TRAIL/Apo2L-based combinatorial regimens for the treatment of colorectal cancers.     

Chloroquine activates the p53 pathway and induces apoptosis in human glioma cells

Glioblastoma is the most common malignant brain tumor in adults. The currently available treatments offer only a palliative survival advantage and the need for effective treatments remains an urgent priority. Activation of the p53 growth suppression/apoptotic pathway is one of the promising strategies in targeting glioma cells. We show that the quinoline derivative chloroquine activates the p53 pathway and suppresses growth of glioma cells in vitro and in vivo in an orthotopic (U87MG) human glioblastoma mouse model. Induction of apoptosis is one of the mechanisms underlying the effects of chloroquine on suppressing glioma cell growth and viability. siRNA-mediated downregulation of p53 in wild-type but not mutant p53 glioblastoma cells substantially impaired chloroquine-induced apoptosis. In addition to its p53-activating effects, chloroquine may also inhibit glioma cell growth via p53-independent mechanisms. Our results clarify the mechanistic basis underlying the antineoplastic effect of chloroquine and reveal its therapeutic potential as an adjunct to glioma chemotherapy.     

Curcumin causes superoxide anion production and p53-independent apoptosis in human colon cancer cells

Curcumin from the rhizome of the Curcuma longa plant has chemopreventative activity and inhibits the growth of neoplastic cells. Since p53 has been suggested to be important for anticancer activity by curcumin, we investigated curcumin-induced cytotoxicity in cultures of p53^+/+ and p53^−/− HCT-116 colon cancer cells, as well as mutant p53 HT-29 colon cancer cells. Curcumin killed wild-type p53 HCT-116 cells and mutant p53 HT-29 cells in a dose- and time-dependent manner. In addition, curcumin-treated p53^+/+ HCT-116 cells and mutant p53 HT-29 cells showed upregulation of total and activated p53, as well as increased expression of p53-regulated p21, PUMA (p53 upregulated modulator of apoptosis), and Bax; however, an equivalent cytotoxic effect by curcumin was observed in p53^+/+ and p53^−/− HCT-116 cells, demonstrating that curcumin-induced cytotoxicity was independent of p53 status. Similar results were obtained when the cytotoxic effect of curcumin was assessed in wild-type p53 HCT-116 cells after siRNA-mediated p53 knockdown. Chromatin condensation, poly (ADP-ribose) polymerase-1 cleavage and reduced pro-caspase-3 levels in curcumin-treated p53^+/+ and p53^−/− HCT-116 cells suggested that curcumin caused apoptosis. In addition, exposure to curcumin resulted in superoxide anion production and phosphorylation of oxidative stress proteins in p53^+/+ and p53^−/− HCT-116 cells. Collectively, our results indicate that, despite p53 upregulation and activation, curcumin-induced apoptosis in colon cancer cells was independent of p53 status and involved oxidative stress. Curcumin may therefore have therapeutic potential in the management of colon cancer, especially in tumors that are resistant to conventional chemotherapy due to defects in p53 expression or function.     

P14ARF sensitizes human osteosarcoma cells to cisplatin-induced apoptosis in a p53-independent manner

The tumor suppressor p14ARF, encoded by the INK4a/ARF locus, is often disrupted in human cancers, p14ARF triggers cell cycle arrest and sensitizes cells to apoptosis in the presence of collateral signals. To investigate the role of p14ARF in chemotherapeutic drugs-induced apoptosis, p14ARF was overexpressed by stable transfection in human osteosarcoma cell lines, U2OS (p53-wt/p14ARF-null) and MG63 (p53-mt/p14ARF-null). The results showed that ectopic p14ARF sensitized both cell lines to cisplatin-induced cytotoxicity and apoptosis. This sensitization of cisplatin-induced apoptosis was associated with upregulation of p53, Bax and p21 in U2OS cells. Conversely, such a result was not observed in MG63 cells. Moreover, the sensitization of cisplatin-induced cytotoxicity in U2OS cells was unaltered by p53 siRNA. Together, we show here p14ARF sensitizes human osteosarcoma cells to cisplatin-induced apoptosis in a p53-independent manner. Proper combinations of p14ARF gene transfer and conventional chemotherapy may be a valuable strategy in human osteosarcoma treatment.