Neobavaisoflavone sensitizes apoptosis via the inhibition of metastasis in TRAIL-resistant human glioma U373MG cells

Aims

Neobavaisoflavone (NBIF), an isoflavone isolated from Psoralea corylifolia (Leguminosae), has striking anti-inflammatory and anti-cancer effects. NBIF inhibits the proliferation of prostate cancer in vitro and in vivo.

Main methods

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a key endogenous molecule that selectively induces apoptosis in cancer cells with little or no toxicity in normal cells. However, some cancer cells, including U373MG cells, are resistant to TRAIL-mediated apoptosis. We demonstrated that the cell viability, migration and invasion assay were used in U373MG glioma cells.

Key findings

In this study, we found that NBIF sensitizes human U373MG glioma cells to TRAIL-mediated apoptosis. Co-treatment of TRAIL and NBIF effectively induced Bid cleavage and activated caspases 3, 8, and 9. Importantly, DR5 expression was upregulated by NBIF. We also observed that the combination NBIF and TRAIL increased expression of BAX. We further demonstrate that NBIF induced TRAIL-mediated apoptosis in human glioma cells by suppressing migration and invasion, and by inhibiting anoikis resistance.

Significance

Taken together, our results suggest that NBIF reduces the resistance of cancer cells to TRAIL and that the combination of NBIF and TRAIL may be a new therapeutic strategy for treating TRAIL-resistant glioma cells.     

The immunoglobulin‐like cell adhesion molecule hepaCAM induces differentiation of human glioblastoma U373‐MG cells

Subsequent to our identification of a novel immunoglobulin‐like cell adhesion molecule hepaCAM, we showed that hepaCAM is frequently lost in diverse human cancers and is capable of modulating cell motility and growth when re‐expressed. Very recently, a molecule identical to hepaCAM (designated as GlialCAM) was found highly expressed in glial cells of the brain. Here, we demonstrate that hepaCAM is capable of inducing differentiation of the human glioblastoma U373‐MG cells. Expression of hepaCAM resulted in a significant increase in the astrocyte differentiation marker glial fibrillary acid protein (GFAP), indicating that hepaCAM promotes glioblastoma cells to undergo differentiation. To determine the relationship between hepaCAM expression level and cell differentiation, we established two U373‐MG cell lines expressing hepaCAM at different levels. The results revealed that high‐level hepaCAM triggered a clear increase in GFAP expression as well as morphological changes characteristic of glioblastoma cell differentiation. Furthermore, high expression of hepaCAM significantly accelerated cell adhesion but inhibited cell proliferation and migration. Concomitantly, deregulation of cell cycle regulatory proteins was detected. Expectedly, the differentiation was noticeably less apparent in cells expressing low‐level hepaCAM. Taken together, our findings suggest that hepaCAM induces differentiation of the glioblastoma U373‐MG cells. The degree of cell differentiation is dependent on the expression level of hepaCAM. J. Cell. Biochem. 107: 1129–1138, 2009. © 2009 Wiley‐Liss, Inc.     

Glaucocalyxin A, a negative Akt regulator, specifically induces apoptosis in human brain glioblastoma U87MG cells

Akt is becoming an attractive target in the development of anti-tumor agents. In the present study, we aimed to discover novel negative Akt regulators against malignant glioma. An Akt regulator screening platform performed in an Akt-GFP overexpression cell line was developed, and natural product library was screened and evaluated using this platform. In addition, the cytotoxic effect of the regulator was detected by MTT assay. Cell apoptosis was assayed by Hoechst 33342 staining and flow cytometry analysis. Afterwards, the apop- totic signaling pathway was investigated by western blot ana- lysis. Glaucocalyxin A, isolated from Rabdosia japonica, was identified as a potent negative regulator of Akt. In human- derived malignant glioma U87MG cells, glaucocalyxin A inhibited Akt phosphorylation, suppressed proliferation, and promoted apoptosis in a dose-dependent manner, but not in normal glial cells. Furthermore, glaucocalyxin A activated caspase-3, decreased BAD phosphorylation, and reduced the expression of X-linked inhibitor of apoptosis protein. Taken together, these results indicated that glaucocalyxin A may become a promising candidate in the treatment of malignant glioma.     

Apoptosis induced by ardipusilloside III through BAD dephosphorylation and cleavage in human glioblastoma U251MG cells

Ardipusilloside III is a saponin newly isolated from Ardisia pusilla A.DC. Since saponins have exhibited broad anti-cancer and pro-apoptotic activity, we investigated the ability of ardipusilloside III to induce apoptosis in human glioblastoma U251MG cells, as well as the involvement of apoptotic signaling pathways. Ardipusilloside III markedly suppressed proliferation of U251MG cells in a time- and dose-dependent manner (P < 0.05, IC₅₀ = 8.2 μg/ml), but did not affect the growth of primary cultures of human astrocytes. Ardipusilloside III-treated U251MG cells underwent typical apoptotic changes. Exposure to a low dose of ardipusilloside III provoked G₂/M-phase cell cycle arrest, which preceded apoptosis characterized by the appearance of cells with sub-G₁ DNA content. However, a higher dose of ardipusilloside III induced apoptosis without first causing cell cycle arrest. In addition, ardipusilloside III exposure resulted in time-dependent BAD dephosphorylation and cleavage as well as activation of caspase-8 and caspase-3. Therefore, both the intrinsic pathway of apoptosis, mediated by BAD dephosphorylation and cleavage, and the extrinisic pathway of apoptosis, mediated by caspase-8 and caspase-3 activation, were involved in ardipusilloside III-induced apoptosis. These data suggest that ardipusilloside III is a reliable candidate for chemotherapeutic treatment of human glioblastomas, and should be investigated further. Hong Lin, Xiang Zhang, Guang Cheng, and Hai-Feng Tang contributed equally to the work.     

Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, functions by inducing apoptosis in human glioblastoma U87MG cells

Malignant glioblastoma is one of the most common malignant tumors in the neurological system. Tubeimoside V (1), a new cyclic bisdesmoside from tubers of Bolbostemma paniculatum, appears to exhibit various biological activities, including antitumor effect, but the function and mechanism of this new agent on glioblastoma cells has not previously been determined. In the present study, we investigated the proliferation change of human glioblastoma U87MG cells exposured to different concentrations (0.9-14.8 microM) of Tubeimoside V (1) for a certain time. The results showed that Tubeimoside V (1) significantly suppressed U87MG cell proliferation in a time- and dose-dependent manner (IC(50) = 3.6 microM). Flow cytometric analysis of DNA in U87MG cells showed that Tubeimoside V (1) induces the prominent appearance of a sub-G1 peak in the cell cycle suggestive of apoptosis. Furthermore, U87MG cells' treatment with Tubeimoside V (1) resulted in nuclear condensation with apoptotic bodies observed by both fluorescence and electron microscopy. The result of annexin V/PI assay showed that phosphatidylserine externalization began after treatment, and then increased in the following 24h. Molecular changes explored through Western-blot staining showed Tubeimoside V (1) decreased the expression levels of Bcl-2 protein and increased the expression levels of Bax protein. The novel findings suggest that the cytotoxic actions of Tubeimoside V (1) toward U87MG cells result from the induction of cell apoptosis. Overall, our data demonstrate that Tubeimoside V (1) is an efficient apoptotic killing agent of glioblastoma cells and suggest that this mechanism may play a critical role in anti-tumor chemotherapy.     

Epigallocatechin-3-gallate suppresses NF-kappaB activation and phosphorylation of p38 MAPK and JNK in human astrocytoma U373MG cells

Epigallocatechin-3-gallate (EGCG) is the major polyphenol component of green tea and is primarily responsible for the green tea effect. EGCG possesses two triphenolic groups in its structure. These groups are reported to be important with respect to anticarcinogenic and antioxidant effects. However, the anti-inflammatory effect of EGCG on Alzheimer's disease (AD) is still not fully understood. In this study, we investigated the effects of EGCG in attenuating the inflammatory response induced by interleukin (IL)-1beta+beta-amyloid (25-35) fragment (Abeta) in human astrocytoma, U373MG cells. EGCG significantly inhibited the IL-1beta+Abeta (25-35)-induced IL-6, IL-8, vascular endothelial growth factor (VEGF) and prostaglandin (PG)E(2) production at 24 h (P<.01). The maximal inhibition rate of IL-6, IL-8, VEGF and PGE(2) production by EGCG was approximately 54.40%, 56.01%, 69.06% and 47.03%, respectively. EGCG also attenuated the expression of cyclooxygenase-2 and activation of nuclear factor-kappaB induced by IL-1beta+Abeta (25-35). We demonstrated that EGCG suppresses IL-1beta+Abeta (25-35)-induced phosphorylation of the mitogen-activated protein kinase p38 and the c-Jun N-terminal kinase. In addition, EGCG induced the expression of mitogen-activated protein kinase phosphatase-1. These results provide new insight into the pharmacological actions of EGCG and its potential therapeutic application to various neurodegenerative diseases such as AD.     

Down-regulation of 14-3-3 zeta sensitizes human glioblastoma cells to apoptosis induction

Strong 14-3-3 zeta protein expression plays an important role in tumorigenesis, including in the maintenance of cell growth, resistance increase, and the prevention of apoptosis. In this study, we focus on two targets: (1) the expression of 14-3-3 zeta in the different grades of human astrocytoma (II–IV), (2) suppression of 14-3-3 zeta protein expression in glioblastoma derived astrocytes by 14-3-3 zeta shRNA lentiviral particles. The tissues of human astrocytoma were provided from 30 patients (ten of each grade of astrocytoma). Control tissues were obtained from the peritumoral brain zone of those patients with glioblastoma. The protein and mRNA expression levels of each astrocytoma grade were assessed via western blotting and RT-PCR, respectively. Results indicated that 14-3-3 zeta was significantly expressed in glioblastoma multiforme (grade IV) and 14-3-3 zeta expression levels enhanced according to the increase of astrocytoma malignancy. In the cellular study for knock down of the 14-3-3 zeta protein, surgical biopsy of glioblastoma was used to isolate primary astrocyte. Astrocytes were transduced with 14-3-3 zeta shRNA or non-targeted shRNA lentiviral particles. Furthermore, reduction of the 14-3-3 zeta protein expression in the astrocytes evaluated through qRT-PCR and western blot after transduction of 14-3-3 zeta shRNA lentiviral particles. Moreover, apoptosis properties, including DNA fragmentation and ratio increase of Bax/Bcl-2 were observed in astrocytes following reduction of 14-3-3 zeta protein expression. Further observation indicated that the mitochondrial pathway through release of cytochorome c and caspase-3 activity was involved in the apoptosis induction. Hence, this study demonstrates a key role of the 14-3-3 zeta protein in tumorigenesis but also indicates that 14-3-3 zeta can be considered as a target for the astrocytoma treatment specially glioblastoma.     

Molecular mechanisms of apoptosis induction by 2-dodecylcyclobutanone,a radiolytic product of palmitic acid,in human lymphoma U937 cells

The irradiation of fat-containing food forms 2-dodecylcyclobutanone (2-DCB) from palmitic acid (PA). In this study, we investigated whether 2-DCB and PA induce apoptosis in human lymphoma U937 cells. We found that cell viability decreased by 2-DCB and apoptosis was induced by 2-DCB and PA. 2-DCB and PA significantly enhanced the formation of intracellular reactive oxygen species (ROS). Apoptosis induced by 2-DCB and PA was strongly prevented by an antioxidant, N-acetyl-l-cysteine. The treatment with 2-DCB and PA resulted in the loss of mitochondrial membrane potential, and Fas, caspase-8 and caspase-3 activation. Pretreatment with a pan-caspase inhibitor (z-VAD) significantly inhibited apoptosis induced by 2-DCB and PA. Moreover, 2-DCB and PA also induced Bax up-regulation, the reduction in Bcl-2 expression level, Bid cleavage and the release of cytochrome c from the mitochondria to the cytosol. In addition, an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) was observed after the treatment with 2-DCB and PA. Our results indicated that intracellular ROS generation, the modulation of the Fas-mitochondrion-caspase-dependent pathway and the increase in [Ca²⁺]_i involved in apoptosis are induced by 2-DCB and PA in U937 cells.     

Calcium response after stimulation by substance P of U373 MG cells: inhibition of store-operated calcium entry by protein kinase C

In this paper we investigate the Ca2+ response after Substance P (SP) stimulation of U373 MG cells. SP is a tachykinin and physiologically acts as a neurotransmitter and neuromodulator in the nervous system, but pathologically triggers malignant glial cells, such as U373 MG, to release cytokines and increase proliferation rate.In this paper we show that SP increases the proliferation rate of U373 MG cells and the intracellular Ca2+ concentration by mobilizing Ca2+ only from thapsigargin-sensitive stores. In fact, Ca2+ entry through store-operated calcium entry (SOCE) channels, which was observed after thapsigargin treatment, was not detected after stimulation by SP. The inhibition of SOCE after SP stimulation must be mediated by protein kinase C (PKC), because it was not observed in the presence of calphostin C (an inhibitor of PKC). Moreover, stimulation by SP-induced membrane potential hyperpolarization. Our results are consistent with the following sequence of events: (i) SP interacts with NK(1) receptors; (ii) fast homologous receptor desensitization occurs; (iii) reuptake by endoplasmic reticulum Ca(2+)-ATPase quantitatively overwhelms the extrusion by plasma membrane Ca2+-ATPase. These results have two important consequences. In U373 MG cells the SOCE does not contribute to the Ca2+ response after SP, and is not necessarily involved in promoting cell proliferation.     

Synergistic anti-cancer mechanisms of curcumin and paclitaxel for growth inhibition of human brain tumor stem cells and LN18 and U138MG cells

Glioblastoma, the deadliest brain tumor in humans, responds poorly to conventional chemotherapeutic agents because of existence of highly chemoresistant human brain tumor stem cells (HBTSC). An effective therapeutic strategy is urgently needed to target HBTSC as well as other glioblastoma cells. We explored synergistic efficacy of a low dose of curcumin (CCM) and a low dose of paclitaxel (PTX) in HBTSC and human glioblastoma LN18 (p53 mutant and PTEN proficient) and U138MG (p53 mutant and PTEN mutant) cells. The highest expression of the cancer stem cell markers aldehyde dehydrogenase 1 (ALDH1) and CD133 occurred in HBTSC when compared with LN18 and U138MG cells. Combination of 20 μM CCM and 10 nM PTX worked synergistically and more effectively than either drug alone in decreasing viability in all cells. Combination of CCM and PTX was highly effective in inducing both morphological and biochemical features of apoptosis. Apoptosis required activation of caspase-8, cleavage of Bid to tBid, increase in Bax:Bcl-2 ratio, and mitochondrial release of cytochrome c, Smac, and apoptosis-inducing factor (AIF). Phosphorylation of Bcl-2 following combination therapy appeared to promote Bax homodimerization and mitochondrial release of pro-apoptotic factors into the cytosol. Increases in activities of cysteine proteases confirmed the completion of apoptotic process. Combination therapy inhibited invasion of cells, reduced expression of survival and proliferation factors and also angiogenic factors, and prevented HBTSC, LN18, and U138MG cells from promoting network formation. Collectively, the combination of CCM and PTX worked as a promising therapy for controlling the growth of HBTSC and other glioblastoma cells.     

Secretory factors of human neuroblastoma (IMR-32) and human glioblastoma (U87MG) cell lines induce neurite outgrowths in PC12 cells

Neurite outgrowth is essential for the communication of the nervous system. The rat Pheochromocytoma (PC12) cells are commonly used in the neuronal cell study. It is well known that exogenous stimuli such as Nerve Growth Factor (NGF) induce neurite outgrowth. In the present study it has been investigated whether or not the conditioned medium from human neuroblastoma cell line (IMR-32) and human glioblastoma cell line (U87MG) may augment neurite outgrowth in PC12 cells. PC12 were cultured with and without conditioned media of IMR-32 and U87MG. The result showed that both the conditioned media induce neurite outgrowth within 48 hr and stops further proliferation of PC12 cells. However no outgrowth was noted in PC12 cells incubated without conditioned medium. In conclusion, it is shown that both the conditioned media (IMR-32 and U87MG) have the potential to induce the neurite outgrowth in the PC12 cells.     

Enhancement of radiation-induced apoptosis of human lymphoma U937 cells by sanazole

Sanazole has been tested clinically as a hypoxic cell radiosensitizer. In this study, we determined whether sanazole enhances the radiation-induced apoptosis of human lymphoma U937 cells. Our results revealed that, compared with 10 mM sanazole or radiation alone, the combination of both resulted in a significant enhancement of apoptosis after 6 h, which was evaluated on the basis of DNA fragmentation, morphological changes, and phosphatidylserine externalization. Sanazole alone enhanced intracellular superoxide and hydrogen peroxide formation, which further increased when the cells were irradiated. Significant enhancement of Fas externalization, loss of mitochondrial membrane potential (MMP), and activation of caspase-3 and caspase-8 were observed after the combined treatment. Moreover, this combination could also enhance Bid activation, reduction of Hsp70 expression level and release of cytochrome c from the mitochondria to the cytosol. An immediate increase in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ) was observed after the combined treatment. These results suggest that the intracellular superoxide and peroxide generated by sanazole might be involved in the enhancement of radiation-induced apoptosis, and that these effects are associated with modulation of the Fas-mitochondria-caspase-dependent pathway, an increase in [Ca²⁺] _i , and a decrease in the Hsp70 expression levels.     

Modulation of plasminogen activator and plasminogen activator inhibitor expression in the human U373 glioblastoma/astrocytoma cell line by inflammatory mediators.

The human U373 glioblastoma/astrocytoma cell line was found to constitutively produce and secrete a plasminogen activator and a plasminogen activator inhibitor. The plasminogen activator was identified as urokinase based on apparent molecular weight, immunoblotting with anti-urokinase antibodies, and Northern blotting with a human urokinase cDNA probe. The inhibitor secreted by U373 cells was found to be related to the PAI-1 molecule based on reactivity with anti-human PAI-1 antibodies, apparent molecular weight, and Northern blot analysis with a human PAI-1 cDNA probe. The expression of both urokinase and the PAI-1-like molecule by U373 cells could be modulated by phorbol myristate acetate or by inflammatory mediators such as interferon-gamma and interleukin-1. In the case of interleukin-1, the alpha form exhibited no detectable effect while the beta form not only elevated inhibitor levels, it also appeared to induce the production of tissue plasminogen activator. Thus, in these cells interleukin-1 beta induces alterations in PA and PAI expression and interleukin-1 alpha does not, even though the two forms are reported to utilize the same cellular receptor.     

Regulation of cadmium-induced apoptosis by PKCdelta in U937 human promonocytic cells

Pulse treatment with cadmium chloride followed by recovery caused apoptosis in U937 human promonocytic cells. In addition, the treatment-induced PKCdelta translocation from cytosol to membrane fraction, which was already detected at 30 min of treatment; and also caused PKCdelta cleavage to give a 41-kDa fragment, which was detected at 3-6 h of recovery, concomitantly with the execution of apoptosis. All these effects were reduced by the PKCdelta-specific inhibitor rottlerin. By contrast, rottlerin did not prevent the cadmium-provoked stimulation of the stress response (as measured by HSP70 expression), nor inhibited the generation of apoptosis by heat-shock, which failed to cause PKCdelta translocation. Cadmium chloride rapidly induced p38(MAPK) activation, which was not affected by rottlerin. By contrast, the p38(MAPK) inhibitor SB203580 reduced PKCdelta translocation and cleavage, indicating that p38(MAPK) activation precedes and regulates PKCdelta activation. It is concluded that PKCdelta mediates apoptosis induction by cadmium ions via early membrane translocation, and also possibly through late kinase proteolytic cleavage and phosphorylation on tyrosine residues.     

Induction of apoptosis in human cervical carcinoma HeLa cells by neoechinulin A from marine-derived fungus Microsporum sp.

Recently, the relationship between apoptosis and cancer has been emphasized and the induction of apoptosis is recognized as one of the key mechanisms of anti-cancer agents. Marine-derived fungi are valuable sources of structurally diverse bioactive compounds with anticancer activity. In the present study, a marine-derived fungus, Microsporum sp. was cultured and a prenylated indole alkaloid, neoechinulin A was isolated from the culture broth extract. Neoechinulin A has shown cytotoxic effect on human cervical carcinoma HeLa cells and its apoptosis induction in HeLa cells was investigated by the expressions of p53, p21, Bax, Bcl-2, Caspase 9, and Caspase 3 proteins. Western blot analysis has revealed that neoechinulin A could induce cell apoptosis through down-regulating of Bcl-2 expression, up-regulating of Bax expression, and activating the caspase-3 pathway. Collectively, these results suggest that neoechinulin A could be a potential candidate in the field of anticancer drug discovery against human cervical cancer.