Induces Caspase Dependent Apoptosis Research Articles

Heliopsis longipes S.F. Blake (Asteraceae) extract causes cell cycle arrest and induces caspase dependent apoptosis against cancer cell lines

Heliopsis longipes is a common spice and a medicinal plant widely used in Mexico and South America due to its mouth anesthetic, analgesic, and anti-inflammatory properties related to its spilanthol content. Previous reports have demonstrated that N-alkylamides exhibit cytotoxic effect against different cancer cell lines; however, the anti-cancer potential of the species remains unknown. Therefore, we investigated the effects of H. longipes extract (HLE) and spilanthol on HeLa, K-562, MCF-7, and HaCaT cells. HLE displayed significative antiproliferative effects and increased the rate of cell apoptosis on all cancer cell lines tested. These effects were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell counting by flow cytometry, cell cycle analysis, Annexin V-FITC/PI activation, and nuclear DAPI staining. Significant growth inhibition was found, with emphasis to K-562 and MCF-7 cells, exhibiting IC50 values of 87.14 and 134.25 μg/mL, respectively, without inducing substantial damage to HaCaT after 48 h of treatment. Spilanthol displayed a more pronounced inhibitory effect on cell lines, maintaining the selectivity index observed for the extract. Furthermore, real-time polymerase chain reaction revealed that HLE activated caspase 3 and caspase 8 after treatment for 48 h. Annexin V-FITC staining to detect early stage of apoptosis and DAPI staining of chromatin substantiated the cytotoxic effects. GC–MS analysis of the extract confirmed spilanthol as the major constituent present (63.94%), followed by other minor alkamides, which could also contribute to the biological effect observed. These results confirm the induction of apoptosis by Heliopsis longipes as well as the potential usefulness of the species and their alkamides as compounds to develop new anti-cancer agents.

A steroidal derivative from Trigonella foenum graecum L. that induces apoptosis in vitro and in vivo

Trigonella foenum graecum L. is a dietary herb used in traditional medicine system. In this study, we investigated the cytotoxicity, antitumor, antimetastatic and antiangiogenic effect of the steroidal compound, ethyl iso-allocholate isolated from T. foenum graecum L. seeds against A549 lung cancer cells in vitro and in vivo. Among all the isolated compounds, the ethyl iso-allocholate rendered the highest cytotoxicity potential. It showed least percentage cell viability in trypan blue assay and lowest nuclei count in hoechst staining. The caspase glo assay and western blot analysis showed the significant caspase 3 cleavage, indicating caspase dependent apoptosis. Consistent with the in vitro data, ethyl iso-allocholate showed highest percentage tumor growth inhibition i.e. 80 ± 5% in zebrafish, equivalent to doxorubicin. It significantly reduced angiogenesis to 5 ± 0.8% (**P < 0.01), compared to negative control group which was 60 ± 2%. The ethyl iso-allocholate showed 55 ± 3% inhibition in liver metastasis. To investigate the safety of the compounds on normal tissues, the percentage mortality was examined. The ethyl iso-allocholate showed zero percent mortality of zebrafish. These results indicate that the steroidal derivative isolated from T. foenum-graecum seeds induces caspase dependent apoptosis in cancer cells and reduces tumor growth, metastasis and angiogenesis in vivo, as well as it is safe on the normal tissues. The in vitro and in vivo anticancer studies suggest that the cytotoxic compound ethyl iso-allocholate has potential application in pharmaceutical industry.

The immunological role of crypt olfactory neurons against neurotropic viruses in teleost fish

Abstract Vertebrate olfactory sensory neurons (OSNs) are located in direct contact with the external environment and therefore they directly exposed to microorganisms such as viruses. Teleost fish have four types of OSNs, including crypt neurons, a cell type with enigmatic function. Crypt neurons express tyrosine receptor kinase A (TrkA)-like molecule and ORA4. In this study, we hypothesized that TrkA expressing crypt neurons are fast sensors of viruses in the olfactory mucosa of fish serving as an ultra-rapid mechanism to elicit local antiviral immune responses. Our results show that, within minutes, nasal delivery of rhadboviruses induces caspase dependent apoptosis in crypt neurons along with neuronal activation in rainbow trout olfactory organ (OO) and olfactory bulb (OB). Induction of chemokine CCL19 and prostaglandin synthase expression was detected 15 minutes after nasal delivery of virus in OO; whereas, in parallel, significant downregulation of CCL19 expression was measured in the OB. Pharmacological blocking of TrkA abolished these responses. CD8+ T cells infiltrated the OO within minutes of nasal viral delivery and this response was abrogated when TrkA was blocked. We identified that the microvasculature surrounding the OB and not the peripheral blood is the source of the infiltrating CD8 T cells suggesting a rapid shunt of these immune cells from the OB microvasculature into the OO. In support, ablation of crypt neurons in zebrafish results in increased susceptibility to rhabdoviral challenge. Combined, our results indicate a novel function for a subset of teleost OSNs, crypt neurons, and the importance of sensory neurons as a first layer of danger detection in vertebrates.

Burkea africana Hook (Caesalpiniaceae) ethanolic extract causes cell cycle arrest at M phase and induces caspase dependent apoptosis

Burkea africana Hook (Caesalpiniaceae) is a small to medium deciduous flat-topped tree up to 20m tall that grows in Western and Southern Africa. Traditionally the bark decoction is used in treatment of fever, inflammation and cancer.Cytotoxic activity of the ethanolic extract was investigated using brine shrimp lethality assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using HeLa cells, cell cycle analysis, histone 3 phosphorylation, Caspase 3 and 8 activity, Annexin-V/FITC activation and mitochondrial membrane depolarization using DiOC6 dye.Burkea africana was not toxic to brine shrimp larvae with an LD50 value of 631μg/mL, while significant growth inhibition was observed in HeLa cells with an IC50 value of 61.4μg/mL. Cell cycle arrest occurred at the G2/M phase, with large increase being observed at the sub-G1 phase, (1.9±0.0% and 3.3±0.0%) control to extract treated cells (14.2±1.1% and 29.8±0.8%) after 24 and 48h. Phosphorylation of histone H3 by the extract (43.86±5.63%) when compared to control (6.35±2.15%) indicates that arrest occurs at the M phase of the cell cycle. Burkea africana activated caspase 3 and caspase 8 after treatment for 24 and 48h. Apoptotic cell death was evident in cells treated with Burkea africana after 30h (15.3±3.5%) when compared with untreated cells (3.5±0.5%). Burkea africana also promoted mitochondrial membrane depolarization as observed in DiOC6 stained cells. These results show for the first time that there are compounds in the extract of Burkea africana with apoptotic effects towards HeLa cells.

Abstract 4182: Statins induce apoptosis in osteosarcoma cells by activation of Ampk and p38-MAPK via suppression of mevalonate pathway

Abstract Osteosarcoma (OS) is the most common, non-hematopoietic, primary malignant bone tumor. OS is characterized by its aggressive local growth and systemic dissemination. Although combination of surgical operation and adjuvant chemotherapy greatly improved the prognosis, more than 20% of patients still cannot get long-term survival. Therefore, novel therapeutic approaches should be expected to be developed. Previously, we developed an OS mouse model by overexpressing c-MYC in bone marrow stromal cells derived from Ink4a/Arf knockout mice. We isolated highly tumorigenic cells (designated AXT cells) from tumors after serial transplantation. Inoculation of AXT cells into syngeneic C57BL/6 mice results in the development of lethal OS with metastatic lesions including lung, which pathologically and clinically mimics human osteoblastic osteosarcoma. To obtain the novel therapeutic agents for OS, we performed drug screening using existing drug collections and found that statins strongly suppressed AXT cell growth and migration in vitro. Simvastatin induces caspase dependent apoptosis, which can be completely rescued by the supplement of mevalonate and geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate, suggesting that OS cells are susceptible to the inhibition of the mevalonate pathway. As the responsible effectors, we found treatment of simvastatin drives RhoA-GTP translocation from cell membrane to cytosol as well as the disruption of interaction between RhoA and Rho-GDI, indicating that, in spite of the accumulation of RhoA-GTP in OS cells, RhoA cannot work properly, which appears to affect cell motility and anchorage dependent survival. As downstream signaling of RhoA, p38-MAPK and AMPK are strongly activated by simvastatin treatment. Activation of p38-MAPK is not accompanied by the ROS production. The specific inhibitor for p38-MAPK or AMPK can rescue this activation as well as the simvastatin-induced cell death, respectively, suggesting that activation of both kinases is responsible for the anti-tumor effect of simvastatin. A single treatment of statins attenuated OS tumor growth in vivo. These findings suggest that the activation of p38-MAPK and AMPK by statins become a potential therapeutic option for OS. Citation Format: Walied Abd-Elghani Kamel, Eiji Sugihara, Sayaka Iwai Yamaguchi, Hiroyuki Nobusue, Kenta Maki, Akihiro Muto, Hideyuki Saya, Takatsune Shimizu. Statins induce apoptosis in osteosarcoma cells by activation of Ampk and p38-MAPK via suppression of mevalonate pathway. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4182.

A novel stereo bioactive metabolite isolated from an endophytic fungus induces caspase dependent apoptosis and STAT-3 inhibition in human leukemia cells

The present study describes the anti-leukemic potential of a novel stereo bioactive secondary metabolite, (R)-5-hydroxy-2-methylchroman-4-one (HMC) isolated from a novel endophytic fungus source (Cryptosporiopsis sp. H2-1, NFCCI-2856), associated with Clidemia hirta. HMC inhibited cell proliferation of different cancer cell lines with IC50 values in the range of 8–55µg/ml. The cytotoxicity window of HMC was 6–12 times lower in normal cells as compared to susceptible leukemic HL-60, MOLT-4 and K-562 cells. It persuades apoptosis through both intrinsic and extrinsic pathways in above leukemic cell lines, which was evident through Hoechst staining, Annexin-V binding, cell cycle analysis, loss of mitochondrial membrane potential (Δψm), release of cytochrome c, Bax, Bid, over-expression of apical death receptors, activation of caspase-3,-8,-9 and PARP (poly ADP ribose polymerase) cleavage. HMC induced caspase dependent apoptosis and robustly attenuate transcription factor, p-STAT-3 in myeloid and lymphoid leukemia cells. The mechanism of HMC arbitrated inhibition of p-STAT-3 was due to the activation of ubiquitin dependent degradation of p-STAT-3. Therefore, our study not only describes the anti-leukemic potential of HMC but also provides insights into how endophytes can be useful in discovery and development of novel anticancer therapeutics.

Influence of gold nanoparticle tagged snake venom protein toxin NKCT1 on Ehrlich ascites carcinoma (EAC) and EAC induced solid tumor bearing male albino mice.

Earlier the conjugation of gold nanoparticle (GNP) and snake venom protein toxin NKCT1 was reported and primary characterization was performed. In the present communication, further characterizations of GNP-NKCT1 were done with SEM, EDS, XRD and Raman spectra for its physio-chemical nature and bonding. SEM showed the formation of gold nanoparticles, whereas EDS and XRD confirmed 60-90% gold nanoparticles in the solution. Raman shift corresponding to (C=O), (N-H), (C-N) confirmed the proper conjugation of GNP with NKCT1. GNP-NKCT1 showed anticancer effect both in vivo and in vitro in EAC cell and antitumor effect in EAC induced mice. In in vivo studies, GNPNKCT1 increased MST 108.30% and decreased viable EAC cell count 51.39%. Fluorescent micrograph showed signs of apoptosis (membrane blebbing, membrane disruption). Decreased level of IL-10 and low incorporation of BrdU showed decreased proliferation of EAC induced by GNP-NKCT1. With upregulation of Bax, down regulation of Bcl2 and increased expression of caspase 3/9, it was confirmed that GNP-NKCT1 induced caspase dependent apoptosis pathway in EAC cell. In in vitro studies, GNP-NKCT1 increased the late apoptotic stage of cell and arrested cell cycle division at G0/G1 state. GNP-NKCT1 also decreased the tumor volume and tumor weight in EAC induced tumor in male albino mice. It inhibited angiogenesis, which was confirmed by lower percentage of expression of VEGF. This study indicated the capability of gold nanoparticles which enhanced the tumor uptake of NKCT1 and also suggested that GNP-NKCT1 might be a good source for anti-carcinoma and anti-tumor agents.

Reversal of boswellic acid analog BA145 induced caspase dependent apoptosis by PI3K inhibitor LY294002 and MEK inhibitor PD98059

PI3K/Akt and ERK pathways are important for growth and proliferation of many types of cancers. Therefore, PI3K inhibitor LY294002 (LY) and MEK1/2 inhibitor PD98059 (PD) are used to sensitize many types of cancer cell lines to chemotherapeutic agents, where AKT and ERK pathways are over activated. However, in this study, we show for the first time that PD could protect the leukemia cells independent of ERK pathway inhibition, besides, we also report a detailed mechanism for antiapoptotic effect of LY in HL-60 cells against the cytotoxicity induced by a boswellic acid analog BA145. Apoptosis induced by BA145 is accompanied by downregulation of PI3K/Akt and ERK pathways in human myelogenous leukemia HL-60 cells, having activating N-Ras mutation. Both LY and PD protected the cells against mitochondrial stress caused by BA145, and reduced the release of cytochrome c and consequent activation of caspase-9. LY and PD also diminished the activation of caspase-8 without affecting the death receptors. Besides, LY and PD also reversed the caspase dependent DNA damage induced by BA145. Further studies revealed that LY and PD significantly reversed the inhibitory effect of BA145 on cell cycle regulatory proteins by upregulating hyperphosphorylated retinoblastoma, pRB (S795) and downregulating p21 and cyclin E. More importantly, all these events were reversed by caspase inhibition by Z-VAD-fmk, suggesting that both LY and PD act at the level of caspases to diminish the apoptosis induced by BA145. These results indicate that inhibitors of PI3K/Akt and ERK pathways can play dual role and act against chemotherapeutic agents.

Abstract 1680: ARHI-induced autophagy enhances chemosensitivity to cisplatin in ovarian cancer cell lines and xenografts.

Abstract Autophagy can protect or kill tumor cells depending upon the context. Our group has found that autophagy and tumor dormancy can be regulated by an imprinted tumor suppressor gene, ARHI (DIRAS3), which is downregulated in 60% of ovarian cancers. Re-expression of ARHI induces autophagy by inhibiting PI3K and mTOR signaling, displacing Bcl-2 from Beclin to form the autophagy initiating complex, inducing ATG4 and decorating the autophagosome membrane, co-localizing with LC3II. Re-expression of ARHI in cell culture produces cell death within 72 hours, whereas re-expression of ARHI in xenografts produces cell growth arrest and tumor dormancy. When ARHI levels are reduced after 6 weeks of induction, xenografts grow promptly. Outgrowth can be delayed if dormant cancers are treated with chloroquine, a functional inhibitor of autophagy, suggesting that autophagy is a survival mechanism in this context. Our current experiments were designed to determine whether autophagic cells could be eliminated more or less readily by treatment with cisplatin with or without chloroquine. In short-term or long-term cell culture, induction of ARHI enhanced the cytotoxic effect of cisplatin and tumor cell killing was further enhanced by the addition of chloroquine. In xenografts, treatment with cisplatin slowed the outgrowth of dormant autophagic ovarian cancer cells, but the addition of chloroquine did not further inhibit xenograft growth. In cell culture, caspase 3/8 release and Anexin V expression on the surface of ovarian cancer cells can account for enhancement of caspase dependent apoptosis observed when autophagic ovarian cancer cells are treated with cisplatin. Treatment of ARHI-induced autophagic ovarian cancer cells with chloroquine and cisplatin downregulated the expression of survivin, XIAP and Bcl-2. Re-expression of ARHI induced caspase dependent apoptosis and autophagic cell death in the absence of cisplatin. ARHI-induced apoptosis and autophagic cell death could be blocked by Z-VAD that inhibits caspase mediated apoptosis and by stable knockdown of ATG5 that inhibits autophagy. In this context, induction of autophagy enhanced platinum toxicity. Additional treatment with chlorooquine produced additional cancer cell cytotoxicity in cell culture, but not in dormant xenografts. These preclinical studies do not support the addition of chloroquine to platinum based chemotherapy for ovarian cancer in the clinic, but do support the association of “autophagic” cell death with apoptosis. Citation Format: Michele N. Washington, Grace Suh, Aaron F. Orozco, Maojie Yang, Yan Wang, Neely Atkinson, Warren Liao, Zhen Lu, Robert Bast. ARHI-induced autophagy enhances chemosensitivity to cisplatin in ovarian cancer cell lines and xenografts. [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 1680. doi:10.1158/1538-7445.AM2013-1680

Induction of Apoptosis in the Rat Bone Marrow Mesenchymal Stem Cells Following Sodium Arsenite Treatment with the Dose Lesser than that Used for Treatment of Malignant Patient.

Arsenic compounds are potent human carcinogen and produce a variety of stress responses in mammalian cells. Recently sodium arsenite has been recommended to be used as anti malignancy drug by American food and drug administration (FDA). In this study, we aimed to determine the apoptosis inducing effect of sodium arsenite on rat bone marrow mesenchymal stem cells exposed in vitro. Cell morphology was studied with the help of Hoechst and propidium iodide as well as with single cell gel electrophoresis(comet assay), TUNEL assay and caspase activity base on immunocytochemistry using commercial kit were considered to study the mechanism of cell death. Our result showed that the sodium arsenite with concentration of 0.1 µM in 36 hr induces caspase dependent apoptosis in rat bone marrow mesenchymal stem cells. This concentration is the lowest level of sodium arsenite to be reported with apoptosis induction ability in stem cells. Since sodium arsenite is used in therapy, more research should be carried out on the effect of this chemical on stem cells, especially MSCs.

PI3K/Akt pathway contributes to development of apoptosis resistance during differentiation of human macrophages by maintaining antiapoptotic Bcl-xL protein expression (48.7)

Abstract Resistance to apoptosis is an important characteristic that human macrophages acquire during differentiation from monocytes. However, the intracellular mechanisms that mediate the development of resistance remain mostly unknown. We have used PMA-treated THP1 cells and MCSF-stimulated primary monocytes to generate human macrophages. By using chemical inhibitors for the PI3K and MAPK pathways, our results show that only pretreatment of cells before differentiation with the PI3K/Akt inhibitor LY294002 induced caspase dependent apoptosis that was associated with decreased expression of antiapoptotic Bcl-xL protein. These results were confirmed by inactivation of this pathway by Akt specific siRNAs. Moreover, this effect was specific if Akt was knocked down before inducing differentiation. Furthermore, Bcl-xL expression was found to be dependent on NFkB in differentiating macrophages. However, NFkB activation was prevented by LY294002 pretreatment, indicating that NFkB is responsible for Bcl-xL expression via PI3K/Akt signaling. Taken together, these results suggest that survival of macrophages is distinctly regulated during and after differentiation. A signaling pathway encompassing PI3K/Akt-mediated activation of NFkB plays a key role in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression.

Abstract 4784: Targeting NF-kappaB pathway with Bay11-7085 leads to inhibition of cell viability and induces apoptosis in primary effusion lymphoma cells

Abstract Primary effusion lymphoma (PEL) is a subtype of non-Hodgkin's B cell lymphoma that mainly grows as a lymphomatous effusion in body cavities and is infected with Kaposi sarcoma-associated herpesvirus (KSVH/HHV8). A number of constitutively activated signaling pathways play critical role in the survival and growth of PEL cells including NF-kB. NF-κB is constitutively activated in a number of cancers including multiple myeloma, burkitt's lymphoma and diffuse large B-cell lymphoma. However, its role in primary effusion lymphoma has not been fully explored. Therefore, in this study, we found that Bay11-7085, an inhibitor of NF-kB pathway inhibit cell viability and induce apoptosis in several PEL cell lines in a dose dependent manner. Such effects of Bay11-7085 result from its ability to in-activate p-IκBα leading to the inability of p65 subunit of NF-κB to enter the nucleus to assert its anti-apoptotic transcriptional activity. Our data demonstrate that Bay11-7085 treatment of PEL cell lines causes de-phosphorylation of Bad protein leading to conformational changes of Bax protein and subsequently translocation from cytosole to mitochondria causing loss of mitochondrial membrane potential with subsequent release of cytochrome c from the mitochondria. Released cytochrome c into the cytosole activates caspases-9 and -3, followed by polyadenosin-5’-diphosphate-ribose polymerase (PARP) cleavage. In addition, pretreatment of PEL cells with zVAD-fmk, a universal inhibitor of caspases prevents caspase-3 activation and abrogates cell death induced by Bay11-7085 treatment of PEL cells strongly suggesting that Bay11-7085-induced apoptosis is caspase dependent. Finally, treatment of PEL cells with Bay11-7085 down-regulates the expression of inhibitor of apoptosis proteins (IAP). Altogether, these data suggest that NF-κB plays an important role in PEL cell survival and targeting this pathway with Bay11-7085 induces caspase dependent apoptosis via disruption of mitochondrial pathway. This raises the possibility that this agent may have a future therapeutic role in PEL and possibly other malignancies with constitutive activation of the NF-κB pathway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4784. doi:10.1158/1538-7445.AM2011-4784

Zalypsis Has Synergistic Effect When Combined with Bortezomib + Dexamethasone through Caspase Dependent and Mainly Independent Mechanisms and through A Potent Induction of DNA Damage

AbstractAbstract 3003During the last years, several novel drugs combinations have changed the outcome of Multiple Myeloma (MM) patients; nevertheless, relapse is still the rule for most of these patients, and, therefore, other drugs with novel mechanisms of action are required. The current research in this topic is usually based on the addition of novel drugs to standard of care combinations in order to try to increase their efficacy. Bortezomib + steroids has turned out to be one of these standards, both for induction therapy before high dose melphalan and also for non-trasplant candidates. Zalypsis is a novel marine compound with remarkable preclinical activity in MM, which is currently being explored in a phase I/II trial. We explored the preclinical efficacy of the triple combination of zalypsis, bortezomib and dexamethasone in different models and investigated its mechanism of action.This combination showed high potency when compared to the respective single agents and double combinations, and, in fact, the analysis by the Calcusyn software, evidenced combination indexes in the highly synergistic range (CI 0.2–0.6). This effect was observed not only in the MM1S cell line, but also in freshly isolated cells from MM patients and, most importantly, in a subcutaneous xenograft of a human plasmocytoma in SCID mice. In this last model, we used suboptimal doses of bortezomib and dexamethasone (0.1 mg/Kg and 1 mg/Kg respectively ip, 5 days per week indefinitely) combined with an active dose of zalypsis (0,75 mg/Kg iv weekly for three total doses). Probably due to the low doses used, the double combination of bortezomib + dexamethasone did not show much effect as compared to the control group, and neither bortezomib nor dexamethasone alone could significantly improve the efficacy of Zalypsis. Nevertheless, when the three drugs were combined together, the tumor volume drastically shrunk with differences highly statistically significant (p<0.001) (Figure 1). These results also translated into an improvement in survival for those mice treated with the triple combination. Median survival (range) of 102 days (85-118) for the triple vs 72 days (61-82) for the best double (Zalypsis + Dexamethasone) (Log Rank < 0.001) (Figure 2). Regarding toxicity, a moderate body weight loss was observed in all mice receiving combinations that included Zalypsis, which completely recovered after stopping treatment with this drug. Two early deaths (out of 10 mice) were also observed in the group of mice assigned to the triple combination group.These results impelled us to investigate the mechanisms relying under the synergy observed. The triple combination induced caspase dependent apoptosis, demonstrated by Annexin V induction and cleavage of caspases 3, 7, 8, 9 and also PARP. By contrast, the activity on the cell cycle was not evident for this combination, indicating a preferential apoptotic effect and not a substantial antiproliferative one. Of note, two mechanisms seem to be specifically associated to the combination of the three agents: 1. A significant release of Endonuclease-G and Cytochrome-C from the mitochondria into the cytosol was observed, suggesting a role for the intrinsic pathway of apoptosis; and 2. One of the most significant changes induced by the combination was an increase in the levels of AIF (apoptosis inducing factor) in the cytosol and nucleus, which is probably indicating the involvement of caspase independent mechanisms. In line with these results, pretreatment with ZVAD was only able to partially rescue from apoptosis, also indicating the potential activation of a dual mechanism: dependent and independent of caspases.Finally, we had previously shown that Zalypsis acts, at least partially, through the induction of DNA damage, based on a p53 dependent mechanism. In this study, the addition of bortezomib and dexamethasone, further increased the H2AX phosphorylation induced by Zalypsis, suggesting a potentiation of the DNA damaging effect; and the combination was also associated with a clear increase in the levels of p53 protein by WB as compared with the agents in monotherapy. The study of the mechanism of action is currently being completed and final results will be presented at the meeting.In summary, the combination of zalypsis + bortezomib + dexamethasone shows high synergy in MM through the activation of different pathways, and these results provide the rationale for its use in relapsed/refractory MM patients. [Display omitted] Disclosures:Cuevas:Pharmamar: Employment. Avilés:Pharmamar: Employment. Pandiella:Pharmamar: Research Funding. San Miguel:Pharmamar: Research Funding; Millenium: Consultancy, Research Funding.

17-DMAG Targets the NF-κB Family of Proteins to Induce Apoptosis in CLL: Clinical Implications of Hsp90 Inhibition

Chronic lymphocytic leukemia (CLL) is a disease of accumulating B-cell lymphocytes for which there is currently no curative therapy. One attractive therapeutic target currently being explored in CLL is Hsp90, a chaperone which stabilizes various client proteins (Akt, Raf, ZAP-70) which are important for survival of CLL cells. Interfering with Hsp90 protein binding to these chaperone proteins leads to their rapid degradation. Our group and others have demonstrated that 17-allylamino-17-demethoxy-geldanamycin (17-AAG) depletes only select chaperone proteins and promotes only modest cytotoxicity in CLL. 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) is a novel Hsp90 inhibitor with improved solubility, bioavailability and cytotoxicity in other cancer cell lines as compared to 17-AAG. We demonstrate that 17-DMAG more potently induces caspase dependent apoptosis of primary CLL cells, as compared to 17-AAG, but does not mediate apoptosis of normal T-cells and NK-cells. In primary CLL cells, 17-DMAG treatment leads to cellular depletion of a broader range of Hsp90 client proteins (Akt, Cdk9, ZAP-70 and IKK). IKK is the activating kinase of the NF-kB family of transcription factors. To validate the down-stream significance of this we used EMSA to confirm that NF-kB is constitutively active in CLL as compared to normal B-cells. We demonstrate that 17-DMAG effectively reduces NF-kB DNA binding and thereby decreases subsequent transcription of known NF-kB target genes (Mcl-1 and XIAP). In addition to the direct role of NF-kB on target genes, NF-kB inhibition indirectly increases Bim transcription through regulation of the FoxO3a transcription factor. Furthermore, the reciprocal regulation transcription of Bcl-2 family members Mcl-1 and Bim are early events that initiate membrane depolarization and activation of the caspase cascade, leading to 17-DMAG induced cytotoxicity. siRNA experiments to elucidate the direct importance of Bim to 17-DMAG treatment are ongoing at this time. In conclusion, our data demonstrate that the Hsp90 inhibitor 17-DMAG represents a novel multi-targeted inhibitor of several critical kinases, transcription factors and anti-apoptosis genes relevant to CLL survival. Given its oral formulation, which allows administration of 17-DMAG by continuous dosing and uninterrupted inhibition of Hsp90, initiation of phase II clinical trials in CLL that include detailed pharmacodynamic studies monitoring NF-kB target genes are indicated.This work is supported by the Leukemia and Lymphoma Society of America and the D. Warren Brown Foundation.

Zoledronic acid induces caspase dependent apoptosis in renal cancer cell lines

Zoledronic acid induces caspase dependent apoptosis in renal cancer cell lines

Apoptotic death induced by the cyclophosphamide analogue mafosfamide in human lymphoblastoid cells: Contribution of DNA replication, transcription inhibition and Chk/p53 signaling

Cyclophosphamide is one of the most often used anticancer drugs. Although DNA interstrand cross-links are considered responsible for its cytotoxicity, the mechanism of initiation and execution of cell death is largely unknown. Using the cyclophosphamide analogue mafosfamide, which does not need metabolic activation, we show that mafosfamide induces apoptosis dose and time dependently in lymphoblastoid cells, with clearly more apoptosis in p53 wt cells. We identified two upstream processes that initiate apoptosis, DNA replication blockage and transcriptional inhibition. In lymphoblastoid cells, wherein DNA replication can be switched off by tetracycline, proliferation is required for inducing apoptosis at low dose mafosfamide. At high dose, transcriptional inhibition also contributes to cell death. The RNA synthesis inhibitor α-amanitin induced similar to mafosfamide more apoptosis in p53 wt than in p53 mt cells. In combination with mafosfamide, however, α-amanitin had no additive effect. Mafosfamide caused p53 stabilization by phosphorylation of Ser15, 20 and 37, and activation of ATM/ATR and Chk1/Chk2. Inhibition of ATM/ATR, PI3-kinase and Chk1/Chk2 by CGK733, wortmannin and DBH, respectively, attenuated the apoptotic response in p53 wt but not p53 mt cells. Mafosfamide induced caspase dependent apoptosis and, for low dose treated cells, caspases were preferentially activated in the S-phase, whereas at high dose caspases were activated in all cell cycle stages. These data support the conclusion that at low dose level of mafosfamide, DNA replication blockage is the dominant apoptosis-inducing event, while at high dose, transcriptional inhibition comes into play. The data provide a mechanistic explanation of why cyclophosphamide applied at therapeutic doses preferentially kills replicating tumor cells.