Natural diterpenes from coffee,cafestol and kahweol induce apoptosis through regulation of specificity protein 1 expression in human malignant pleural mesothelioma

Background

Malignant pleural mesothelioma (MPM) is a highly aggressive cancer with a very poor prognosis. Several clinical studies such as immunotherapy, gene therapy and molecular targeting agents have been tried for treatment of malignant mesothelioma, however, there is no application for effective clinical treatment. Coffee has various biological functions such as anti-oxidant, anti-inflammatory, anti-mutagenic and anti-carcinogenic activities. The therapeutic activities of the bioactive compounds in coffee was sugested to influence intracellular signaling of MPM. Regarding to the cancer-related functions, In this study, suppression of Sp1 protein level followed by induction of MSTO-211H cell apoptosis by cafestol and kahweol were investigated in oreder to determine Sp1''s potential as a significant target for human MPM therapy as well.

Methods

Cells were treated separately with final concentration of cafestol and kahweol and the results were analyzed by MTS assay, DAPI staining, PI staining, luciferase assay, RT-PCR, and immunoblotting.

Results

Viability of MSTO-211H and H28 cells were decreased, and apoptotic cell death was increased in MSTO-211H as a result of cafestol and kahweol treatment. Cafestol and kahweol increased Sub-G₁ population and nuclear condensation in MSTO-211H cells. Roles of Sp1 in cell proliferation and apoptosis of the MSTO-211H cells by the Sp1 inhibitor of Mithramycin A were previously confirmed. Cafestol and kahweol significantly suppressed Sp1 protein levels. Kahweol slightly attenuated Sp1 mRNA, while Cafestol did not affect in MSTO-211H cells. Cafestol and kahweol modulated the promoter activity and protein expression level of the Sp1 regulatory genes including Cyclin D1, Mcl-1, and Survivin in mesothelioma cells. Apoptosis signaling cascade was activated by cleavages of Bid, Caspase-3, and PARP with cafestol and by upregulation of Bax, and downregulation of Bcl-_xl by kahweol.

Conclusions

Sp1 can be a novel molecular target of cafestol and kahweol in human MPM.     

BAI,A novel cyclin‐dependent kinase inhibitor induces apoptosis in A549 cells through activation of caspases and inactivation of Akt

Previously, we have synthesized a novel cyclin‐dependent kinase (CDK) inhibitor, 2‐[1,1′biphenyl]‐4‐yl‐N‐[5‐(1,1‐dioxo‐1λ⁶‐isothiazolidin‐2‐yl)‐1H‐indazol‐3‐yl]acetamide (BAI) and reported its anti‐cancer activity in head and neck cancer cells. In this study, we further evaluated the effect of BAI on growth of various human cancer cell lines, including A549 (nonsmall cell lung cancer), HCT116 (colon), and Caki (kidney). Profoundly, results of XTT and clonogenic assays demonstrated that BAI at nanomolar concentrations (20–60 nM) inhibited growth of A549, HCT116, and Caki cells, suggesting the anti‐cancer potency. We show that BAI induced a dose‐dependent apoptotic cell death in these human cancer cells, as measured by fluorescence‐activated cell sorting (FACS). Interestingly, further biochemical analysis showed that treatment with BAI at 20 nM induced apoptosis in A549 cells in association with activation of caspases, cleavage of phospholipase C‐γ1 (PLC‐γ1), and inhibition of Akt in A549 cells. Importantly, pharmacological inhibition study revealed that pretreatment with z‐VAD‐fmk, a pan caspase inhibitor strongly blocked the BAI‐induced apoptosis in A549 cells. Transfection analysis with Akt cDNA encoding constitutively active Akt further addressed the significance of Akt inhibition in the BAI‐induced apoptosis in A549 cells. Notably, disruption of the PI3K/Akt pathway by LY294002, a PI3K/Akt inhibitor potentiated apoptosis in A549 cells by BAI at a subcytotoxic concentration. These findings collectively suggest that BAI potently inhibits growth of A549, HCT116, and Caki cells, and that the BAI‐induced apoptosis in A549 cells is associated with activation of caspases, and inhibition of Akt. J. Cell. Biochem. 114: 282–293, 2013. © 2012 Wiley Periodicals, Inc.     

Mcl-1 is a key regulator of apoptosis resistance in Chlamydia trachomatis-infected cells

Chlamydia are obligate intracellular bacteria that cause variety of human diseases. Host cells infected with Chlamydia are protected against many different apoptotic stimuli. The induction of apoptosis resistance is thought to be an important immune escape mechanism allowing Chlamydia to replicate inside the host cell. Infection with C. trachomatis activates the Raf/MEK/ERK pathway and the PI3K/AKT pathway. Here we show that inhibition of these two pathways by chemical inhibitors sensitized C. trachomatis infected cells to granzyme B-mediated cell death. Infection leads to the Raf/MEK/ERK-mediated up-regulation and PI3K-dependent stabilization of the anti-apoptotic Bcl-2 family member Mcl-1. Consistently, interfering with Mcl-1 up-regulation sensitized infected cells for apoptosis induced via the TNF receptor, DNA damage, granzyme B and stress. Our data suggest that Mcl-1 up-regulation is primarily required to maintain apoptosis resistance in C. trachomatis-infected cells.     

Cyclooxygenase-2 inducing Mcl-1-dependent survival mechanism in human lung adenocarcinoma CL1.0 cells. Involvement of phosphatidylinositol 3-kinase/Akt pathway

Cyclooxygenase 2 (COX-2) has been reported to be commonly expressed in advanced stages of human lung adenocarcinoma. In this study, the COX-2 constitutive expression vector was transfected into a human lung adenocarcinoma cell line CL1.0 and several clones were obtained which stably expressed COX-2. These COX-2-overexpressed clones demonstrated remarkable resistance to apoptosis induced by Ultraviolet B (UVB) irradiation, vinblastine B (VBL) cell lymphoma-2 (Bcl-2), or other anti-cancer drugs. To understand how COX-2 prevents apoptosis, the investigators examined the expression level of Bcl-2 family members. Mcl-1, but not other Bcl-2 members, was significantly up-regulated by COX-2 transfection or prostaglandin E(2) (PGE(2)) treatment. Treatment of COX-2-overexpressed cells (cox-2/cl.4) with two specific COX-2 inhibitors, NS-398 and celecoxib, caused an effective reduction of the increased level of Mcl-1. These data suggest that the expression level of Mcl-1 is tightly regulated by COX-2. Moreover, transfection of cox-2/cl.4 cells with antisense Mcl-1 enhanced apoptosis induced by UVB irradiation, revealing that Mcl-1 plays a crucial role in cell survival activity mediated by COX-2. Furthermore, COX-2 transfection or PGE(2) treatment evidently activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Inhibition of the PI3K pathway by LY294002 or wortmannin effectively attenuated the increased level of Mcl-1 induced by COX-2 or PGE(2). Blocking the PI3K activity with a dominant-negative vector, DN-p85, also greatly diminished the level of Mcl-1 and enhanced UVB-elicited cell death in cells transfected by COX-2. In a similar way, LY294002 inhibited cell survival and Mcl-1 level in PGE(2)-treated CL1.0 cells. These findings suggest that COX-2 promotes cell survival by up-regulating the level of Mcl-1 by activating the PI3K/Akt-dependent pathway.     

The novel phospholipase C activator, <Emphasis Type="Italic">m</Emphasis>-3M3FBS,induces apoptosis in tumor cells through caspase activation,down-regulation of XIAP and intracellular calcium signaling

We investigated the effect of the novel phospholipase C activator, m-3M3FBS, on the apoptosis of human renal Caki cancer cells. Treatment with m-3M3FBS induced apoptosis of Caki cells, which was accompanied by accumulation of sub-G1 phase and DNA fragmentation. We found that induction of apoptosis is a common response of several cancer cell types to m-3M3FBS treatment. Overexpression of Bcl-2 and c-FLIPs fails to block m-3M3FBS-induced apoptosis. However, ectopic expression of XIAP partly inhibits m-3M3FBS-induced apoptosis in Caki cells. m-3M3FBS-induced apoptosis appeared to involve the XIAP down-regulation and caspase activation. m-3M3FBS also induced the expression of a potential proapoptotic gene, C/EBP homologous protein (CHOP), however, suppression of CHOP expression by small interfering RNA did not abrogate the m-3M3FBS-induced apoptosis. In addition, inhibition of phospholipase C (PLC) or chelation of intracellular calcium prevented m-3M3FBS-induced apoptosis in Caki cells, suggesting that the involvement of PLC pathway and intracellular calcium signaling on the apoptosis in m-3M3FBS-treated Caki cells. Collectively, our present results suggest that m-3M3FBS-induced apoptosis in Caki cells may result from the activation of caspase, down-regulation of XIAP and intracellular Ca²⁺ release pathway and that m-3M3FBS treatment might overcome the anti-apoptotic effect of Bcl-2 or c-FLIPs in cancer cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Eun Mi Jung and Tae-Jin Lee contributed equally to this work.     

The coffee diterpene kahweol sensitizes TRAIL-induced apoptosis in renal carcinoma Caki cells through down-regulation of Bcl-2 and c-FLIP

Kahweol, a coffee-specific diterpene, found in the beans of Coffea arabica, has potent anti-carcinogenic, anti-tumor, and anti-inflammatory properties. TRAIL is a potential anti-cancer compound that induces apoptosis in a wide variety of cancer cells, but not in most normal human cell types. In the present study, we show that kahweol sensitizes human renal cancer cells, but not normal human mesangial cells, to TRAIL-mediated apoptosis. Moreover, treatment with a combination of kahweol and TRAIL induces significant apoptosis in various cancer cell types, thus presenting an attractive novel strategy for cancer treatment. Our experiments show that treatment with a combination of kahweol and TRAIL-induced apoptosis, and stimulated of DEVDase activity, DNA fragmentation, and cleavage of PARP, which was prevented by pretreatment with z-VAD, indicative of cell death via a caspase-dependent pathway. Kahweol-induced down-regulation of Bcl-2 and ectopic expression of Bcl-2 led to attenuation of kahweol plus TRAIL-mediated apoptosis, indicative of Bcl-2 involvement in the apoptotic process. In addition, the c-FLIP and caspase signal pathways seem to play a crucial role in apoptosis triggered by the combination of kahweol and TRAIL in Caki cells. Our results collectively demonstrate that down-regulation of Bcl-2 and c-FLIP contributes to the sensitizing effect of kahweol on TRAIL-mediated apoptosis in cancer cells.     

The coffee diterpene kahweol induces apoptosis in human leukemia U937 cells through down-regulation of Akt phosphorylation and activation of JNK

Kahweol, the coffee-specific diterpene, has been reported for its tumor cell growth inhibitory activity and anti-carcinogenic activity. The mechanism by which kahweol initiates apoptosis remains poorly understood. In the present study, we investigated the effect of kahweol on the apoptotic pathway in U937 human promonocytic cells. We show that kahweol induces apoptosis in association with the activation of caspase 3 and cytochrome c release from the mitochondria to the cytosol, as well as down-regulation of anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1 and XIAP). Kahweol altered the phosphorylation state of members of the MAPKs and Akt. Ectopic expression of Bcl-2 or constitutive active Akt (myr-Akt) in U937 cells attenuates kahweol-induced apoptosis. In addition, we have also shown that JNK and Akt signal pathway plays a crucial role in kahweol-induced apoptosis in U937 cells. Taken together, our results show the activity of kahweol to modulate multiple components in apoptotic response of human leukemia cells and raise the possibility a novel therapeutic strategy in hematological malignancies.     

Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-x_L, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.     

Chrysin-induced apoptosis is mediated through caspase activation and Akt inactivation in U937 leukemia cells

Chrysin is a natural, biologically active compound extracted from many plants, honey, and propolis. It possesses potent anti-inflammation, anti-cancer, and anti-oxidation properties. The mechanism by which chrysin initiates apoptosis remains poorly understood. In the present report, we investigated the effect of chrysin on the apoptotic pathway in U937 human promonocytic cells. We show that chrysin induces apoptosis in association with the activation of caspase 3 and that Akt signal pathway plays a crucial role in chrysin-induced apoptosis in U937 cells. Furthermore, we have shown that inhibition of Akt phosphorylation in U937 cells by the specific PI3K inhibitor, LY294002 significantly, enhanced apoptosis. Overexpression of a constitutively active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase 3, and PLC-gamma1 cleavage by chrysin. Together, these findings suggest that the Akt pathway plays a major role in regulating the apoptotic response of human leukemia cells to chrysin and raise the possibility that combined interruption of chrysin and PI3K/Akt-related pathways may represent a novel therapeutic strategy in hematological malignancies.     

Apoptosis of CTLL-2 cells induced by an immunosuppressant, ISP-I, is caspase-3-like protease-independent

In our previous study, the sphingosine-like immunosuppressant ISP-1 was shown to induce apoptosis in the mouse cytotoxic T cell line CTLL-2. In this study, we characterized the ISP-1-induced apoptotic pathway. Although caspase-3-like protease activity increases concomitantly with ISP-1-induced apoptosis in CTLL-2 cells, the apoptosis is not inhibited by caspase-3-like protease inhibitors, i.e. DEVD-cho and z-DEVD-fmk. In contrast, sphingosine-induced apoptosis in CTLL-2 cells is caspase-3-like protease-dependent. A caspase inhibitor with broad specificity, z-VAD-fmk, protects cells from apoptosis induced by ISP-1, indicating that ISP-1-induced apoptosis is dependent on caspase(s) other than caspase-3. Overexpression of Bcl-2 or Bcl-xL suppresses the apoptosis induced by ISP-1, although sphingosine-induced apoptosis is not efficiently inhibited by Bcl-2. Finally, ISP-1-induced mitochondrial depolarization, which is thought to be a checkpoint dividing the apoptotic pathway into upstream and downstream stages, is not inhibited by DEVD-cho, but is inhibited by z-VAD-fmk. These data suggest that a pathway dependent on caspase(s) other than caspase-3 is involved upstream of mitochondrial depolarization in ISP-1-induced apoptosis.     

Angelicin induces apoptosis through intrinsic caspase-dependent pathway in human SH-SY5Y neuroblastoma cells

Angelicin is structurally related to psoralens, a well-known chemical class of photosensitizers used for its antiproliferative activity in treatment of different skin diseases. To verify the activity of angelicin, we employed human SH-SY5Y neuroblastoma cells to investigate its cytotoxicity, although its mechanism of action has not yet been fully elucidated. Here, we examined the cellular cytotoxicity of angelicin by cell viability assay, DNA fragmentation by DNA ladder assay, and activation of caspases and Bcl-2 family proteins by western blot analyses. The results of our investigation suggest that angelicin increased cellular cytotoxicity in a dose- and time-dependent manner with IC(50) of 49.56?μM at 48?h of incubation. In addition, angelicin dose-dependently downregulated the expression of anti-apoptotic proteins including Bcl-2, Bcl-xL, and Mcl-1 suggesting the involvement of the intrinsic mitochondria-mediated apoptotic pathway which did not participate in Fas/FasL-induced caspase-8-mediated extrinsic, MAP kinases, and PI3K/AKT/GSK-3β pathway. Furthermore, we clarified the dose-dependent upregulation of caspase-9 and caspase-3 which indicated that angelicin-induced apoptosis is mediated primarily through the intrinsic caspase-mediated pathway. In particular, the caspase-3 inhibitor, DEVD-fmk, induced a reduction in angelicin-induced cytotoxicity which confirmed that the intrinsic caspase-dependent pathway during this apoptosis which did not prevent cytotoxicity using MAP kinases and GSK-3 inhibitor. Taken together, our data shows that angelicin is an effective apoptosis-inducing natural compound of human SH-SY5Y neuroblastoma cells which suggests that this compound may have a role in future therapies for human neuroblastoma cancer.     

Cooperative regulation of Mcl-1 by Janus kinase/stat and phosphatidylinositol 3-kinase contribute to granulocyte-macrophage colony-stimulating factor-delayed apoptosis in human neutrophils

Polymorphonuclear neutrophils (PMN) are phagocytic cells constitutively programmed for apoptotic cell death. Exposure to GM-CSF delays apoptosis as measured by annexin-V staining and cell morphological change. We found that STAT5B, STAT1, and STAT3 DNA-binding activity was induced by GM-CSF. We also detected activation of the phosphatidylinositol 3-kinase (PI 3-kinase) pathway after GM-CSF treatment which was inhibited by treatment with the PI 3-kinase inhibitors, wortmannin and LY294002. We investigated whether STAT or PI 3-kinase activity was necessary for the pro-survival response of GM-CSF in PMN. Exposure of PMN to GM-CSF in the presence of either AG-490, antisense STAT3 oligonucleotides, or wortmannin resulted in a partial inhibition of GM-CSF-mediated pro-survival activity. GM-CSF induced a time-dependent increase in the mRNA and protein expression of the anti-apoptotic Bcl-2-family protein, Mcl-1. We examined the hypothesis that Janus kinase/STAT and PI 3-kinase regulation of Mcl-1 contributed to GM-CSF-delayed apoptosis. Using either AG-490 or wortmannin alone, we observed a dose-dependent inhibition of GM-CSF-induced Mcl-1 expression. Using suboptimal doses of AG-490 and wortmannin, we found that both drugs together had an additive effect on delayed apoptosis and Mcl-1 expression. These data suggest that cooperative regulation of Mcl-1 by the Janus kinase/STAT and PI 3-kinase pathways contribute to GM-CSF-delayed apoptosis.     

3,3'-Diindolylmethane exhibits antileukemic activity in vitro and in vivo through a Akt-dependent process

3,3'-Diindolylmethane (DIM), one of the active products derived from Brassica plants, is a promising antitumor agent. The present study indicated that DIM significantly induced apoptosis in U937 human leukemia cells in dose- and time-dependent manners. These events were also noted in other human leukemia cells (Jurkat and HL-60) and primary human leukemia cells (AML) but not in normal bone marrow mononuclear cells. We also found that DIM-induced lethality is associated with caspases activation, myeloid cell leukemia-1 (Mcl-1) down-regulation, p21(cip1/waf1) up-regulation, and Akt inactivation accompanied by c-jun NH2-terminal kinase (JNK) activation. Enforced activation of Akt by a constitutively active Akt construct prevented DIM-mediated caspase activation, Mcl-1 down-regulation, JNK activation, and apoptosis. Conversely, DIM lethality was potentiated by the PI3K inhibitor LY294002. Interruption of the JNK pathway by pharmacologic or genetic approaches attenuated DIM-induced caspases activation, Mcl-1 down-regulation, and apoptosis. Lastly, DIM inhibits tumor growth of mouse U937 xenograft, which was related to induction of apoptosis and inactivation of Akt, as well as activation of JNK. Collectively, these findings suggest that DIM induces apoptosis in human leukemia cell lines and primary human leukemia cells, and exhibits antileukemic activity in vivo through Akt inactivation and JNK activation.     

Granulocyte macrophage colony-stimulating factor signaling and proteasome inhibition delay neutrophil apoptosis by increasing the stability of Mcl-1

Human neutrophils normally have a very short half-life and die by apoptosis. Cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) can delay this apoptosis via increases in the cellular levels of Mcl-1, an anti-apoptotic protein of the Bcl-2 family with a rapid turnover rate. Here we have shown that inhibition of the proteasome (a) decreases the rate of Mcl-1 turnover within neutrophils and (b) significantly delays apoptosis. This led us to determine whether GM-CSF could enhance neutrophil survival by altering the rate of Mcl-1 turnover. Addition of GM-CSF to neutrophils enhanced Mcl-1 stability and delayed apoptosis by signaling pathways requiring PI3K/Akt and p44/42 Erk/Mek, because inhibitors of these pathways completely abrogated the GM-CSF-mediated effect on both Mcl-1 stability and apoptosis delay. Conversely, induction of Mcl-1 hyperphosphorylation by the phosphatase inhibitor, okadaic acid, significantly accelerated both Mcl-1 turnover and apoptosis. Neither the calpain inhibitor, carbobenzoxy-valinyl-phenylalaninal, nor the pan caspase inhibitor, benzyloxycarbonyl-VAD-fluoromethylketone, had any effect on Mcl-1 stability under these conditions. These observations indicate that profound changes in the rate of neutrophil apoptosis following cytokine signaling occur via dynamic changes in the rate of Mcl-1 turnover via the proteasome.