Gamma-tocotrienol induces apoptosis and autophagy in prostate cancer cells by increasing intracellular dihydrosphingosine and dihydroceramide

Although cell-based studies have shown that γ-tocotrienol (γTE) exhibits stronger anticancer activities than other forms of vitamin E including γ-tocopherol (γT), the molecular bases underlying γTE-exerted effects remains to be elucidated. Here we showed that γTE treatment promoted apoptosis, necrosis and autophagy in human prostate PC-3 and LNCaP cancer cells. In search of potential mechanisms of γTE-provoked effects, we found that γTE treatment led to marked increase of intracellular dihydroceramide and dihydrosphingosine, the sphingolipid intermediates in de novo sphingolipid synthesis pathway but had no effects on ceramide or sphingosine. The elevation of these sphingolipids by γTE preceded or coincided with biochemical and morphological signs of cell death and was much more pronounced than that induced by γT, which accompanied with much higher cellular uptake of γTE than γT. The importance of sphingolipid accumulation in γTE-caused fatality was underscored by the observation that dihydrosphingosine and dihydroceramide potently reduced the viability of both prostate cell lines and LNCaP cells, respectively. In addition, myriosin, a specific inhibitor of de novo sphingolipid synthesis, counteracted γTE-induced cell death. In agreement with these cell-based studies, γTE inhibited LNCaP xenograft growth by 53% (p < 0.05), compared to 33% (p = 0.07) by γT, in nude mice. These findings provide a molecular basis of γTE-stimulated cancer cell death and support the notion that elevation of intracellular dihydroceramide and dihydrosphingosine is likely a novel anticancer mechanism.     

Geraniol induces cooperative interaction of apoptosis and autophagy to elicit cell death in PC-3 prostate cancer cells

Geraniol, an acyclic dietary monoterpene, suppresses prostate cancer growth and enhances docetaxel chemosensitivity in cultured cell or xenograft tumor models. However, the mechanisms of the geraniol action against prostate cancer are largely unknown. In this study, we investigated the cellular and molecular mechanisms of geraniol-induced cell death in PC-3 prostate cancer cells. Among the examined structurally and functionally similar monoterpenes, geraniol potently induced apoptosis and autophagy. Although independent processes, apoptosis and autophagy acted as cooperative partners to elicit geraniol-induced cell death in PC-3 cells. At a molecular level, geraniol inhibited AKT signaling and activated AMPK signaling, resulting in mTOR inhibition. Combined treatment of AKT inhibitor and AMPK activator markedly suppressed cell growth compared to either treatment alone. Our findings provide insight into future investigations that are aimed at elucidating the role of apoptosis and autophagy in prostate cancer therapy and at developing anticancer strategies co-targeting AKT and AMPK.     

Resveratrol induces apoptosis via ROS-triggered autophagy in human colon cancer cells

Resveratrol (Res; 3,4',5-trihydroxy-trans-stilbene), which is a polyphenol found in grapes, can block cell proliferation and induce growth arrest and/or cell death in several types of cancer cells. However, the precise mechanisms by which Res exerts anticancer effects remain poorly understood. Res blocked both anchorage-dependent and -independent growth of HT-29 and COLO 201 human colon cancer cells in a dose- and time-dependent manner. Annexin V staining and Western blot analysis revealed that Res induced apoptosis accompanied by an increase in Caspase-8 and Caspase-3 cleavage. In HT-29 cells, Res caused autophagy as characterized by the appearance of autophagic vacuoles by electron microscopy and elevation of microtubule-associated protein 1 light chain 3 (LC3)-II by immunoblotting, which was associated with the punctuate pattern of LC3 detected by fluorescein microscopy. Inhibition of Res-induced autophagy by the autophagy inhibitor 3-methyladenine caused a significant decrease in apoptosis accompanied by decreased cleavage of Casapse-8 and Caspase-3, indicating that Res-induced autophagy was cytotoxic. However, inhibition of Res-induced apoptosis by the pan-caspase inhibitor Z-VAD(OMe)-FMK did not decrease autophagy but elevated LC3-II levels. Interestingly, Res increased the intracellular reactive oxygen species (ROS) level, which correlated to the induction of Casapse-8 and Caspase-3 cleavage and the elevation of LC3-II; treatment with ROS scavenger N-acetyl cysteine diminished this effect. Therefore, the effect of Res on the induction of apoptosis via autophagy is mediated through ROS in human colon cancer cells.     

塞来昔布诱导胃癌SGC-7901细胞凋亡和自噬

目的:观察塞来昔布对人胃癌细胞株SGC-7901凋亡和自噬的影响,并探讨其凋亡的机制。方法:不同浓度塞来昔布处理SGC-7901细胞后,MTT法检测SGC-7901细胞的增殖,TUNEL法检测SGC-7901细胞的凋亡,透射电镜观察SGC-7901细胞超微结构的改变,流式细胞术检测SGC-7901细胞的凋亡率,实时定量荧光PCR法检测SGC-7901细胞中caspase-8和caspase-9 mRNA的表达。结果:塞来昔布时间(24、48、72 h)和剂量(50、75、100、125μmol/L)依赖性抑制SGC-7901细胞的增殖,125μmol/L塞来昔布作用SGC-7901 72 h细胞的增殖抑制率高达(85.6±4.51)%。塞来昔布可诱导SGC-7901细胞凋亡,透射电镜下观察到典型的凋亡小体和自噬体,细胞凋亡率从(2.2±1.32)%上升到(35.7±5.73)%(P<0.01)。塞来昔布作用后,SGC-7901细胞中caspase-8和caspase-9 mRNA表达明显增加,呈时间和剂量依赖性(P<0.05)。结论:塞来昔布通过激活依赖caspase-8的死亡受体途径和依赖caspase-9的线粒体途径诱导胃癌SGC-7901细胞凋亡,同时诱发自噬性细胞死亡。     

Lysosomotropic acid ceramidase inhibitor induces apoptosis in prostate cancer cells

Purpose Alterations in ceramide metabolism have been reported in prostate cancer (PCa), resulting in escape of cancer cells from ceramide-induced apoptosis. Specifically, increased expression of lysosomal acid ceramidase (AC) has been shown in some primary PCa tissues and in several PCa cell lines. To determine if this represents a novel therapeutic target, we designed and synthesized LCL204, a lysosomotropic analog of B13, a previously reported inhibitor of AC Methods Prostate cancer cell lines were treated with LCL204 for varying times and concentrations. Effects of treatment on cytotoxicity, sphingolipid content, and apoptotic markers were assessed. Results Treatment of DU145 PCa cells resulted in increased ceramide and decreased sphingosine levels. Interestingly, LCL204 caused degradation of AC in a cathepsin-dependent manner. We also observed rapid destabilization of lysosomes and the release of lysosomal proteases into the cytosol following treatment with LCL204. Combined, these events resulted in mitochondria depolarization and executioner caspase activation, ultimately ending in apoptosis Conclusions These results provide evidence that treatment with molecules such as LCL204, which restore ceramide levels in PCa cells may serve as a new viable treatment option for PCa.     

桧木醇激活膀胱癌J82细胞自噬诱导细胞凋亡

背景与目的：膀胱癌是我国最常见的泌尿系统恶性肿瘤,近年来发病率逐年上升,严重威胁着人类的健康。本研究旨在探讨桧木醇对人膀胱癌J82细胞增殖、凋亡及自噬的作用,并初步阐明其作用机制。方法：采用CCK-8法检测细胞的增殖活力,采用流式细胞术检测细胞的凋亡状态,采用蛋白[质]印迹法(Western blot)检测cleaved caspase 3、LC3及P62蛋白的表达,转染EGFP-LC3后在激光共聚焦显微镜下观察细胞自噬状态。结果：桧木醇能够显著抑制J82细胞的增殖活力,通过激活caspase途径诱导肿瘤细胞凋亡,Z-VAD-FMK能够部分抑制桧木醇的凋亡诱导作用。桧木醇能够激活J82细胞发生自噬,上调LC3蛋白的表达,下调P62蛋白的表达。3-MA抑制自噬之后能够部分逆转桧木醇的抗肿瘤作用。结论：桧木醇可以显著抑制J82细胞增殖并通过过度激活自噬促进膀胱癌细胞凋亡。     

Rottlerin induces autophagy and apoptosis in prostate cancer stem cells via PI3K/Akt/mTOR signaling pathway

Autophagy plays an important role in cellular homeostasis through the disposal and recycling of cellular components. Cancer stem cells (CSCs) play major roles in cancer initiation, progression, and drug resistance. Rottlerin (Rott) is an active molecule isolated from Mallotus philippinensis, a medicinal plant used in Ayurvedic Medicine for anti-allergic and anti-helminthic treatments, demonstrates anticancer activities. However, the molecular mechanisms by which it induces autophagy in prostate CSCs have not been examined. The main objective of the paper was to examine the molecular mechanisms by which Rott induces autophagy in prostate CSCs. Autophagy was measured by the lipid modification of light chain-3 (LC3) and the formation of autophagosomes. Apoptosis was measured by flow cytometer analysis. The Western blot analysis was used to examine the effects of Rott on the expression of PI3K, phosphorylation of Akt, phosphorylation of mTOR, and phosphorylation of AMPK in pros CSCs. RNAi technology was used to inhibit the expression of Beclin-1 and ATG-7. Rott induced the lipid modification of light chain-3 (LC3) and the formation of autophagosomes after 24 h of Rott treatment in prostate CSCs. Rott-treated prostate CSCs induced transition from LC3-I to LC3-II, a hall mark of autophagy. Rott also induced the expression of Atg5, Atg7, Atg12 and Beclin-1 proteins during autophagy. The knock-down of Atg7 and Beclin-1 blocked Rott-induced autophagy. Furthermore, Rott induced AMPK phosphorylation was blocked by 3-MA, Baf and CHX. In addition, inhibition of AMPK expression by shRNA blocked Rott induced autophagy. In conclusion, a better understanding of the biology of autophagy and the pharmacology of autophagy modulators has the potential for facilitating the development of autophagy-based therapeutic interventions for prostate cancer.     

Piplartine induces caspase-mediated apoptosis in PC-3 human prostate cancer cells

The present study examined the anti-proliferative effects of piplartine on the human prostate cancer cell line PC-3. This is the first report demonstrating the piplartine anti-cancer activity toward prostate cancer cell lines, although its precise mechanism of action is still not completely defined. In MTT assays, it preferentially inhibited growth of androgen-independent PC-3 cells in a dose-dependent (3-30 microM) and time-dependent (12-48 h) manner. In PC-3 cells, it showed an IC50 of 15 microM after 24 h of treatment. After a 24-30 microM treatment for 24 h, there were some reduction of cell volume, cell vacuolization, chromatin condensation and increased number of apoptotic cells visible by light and fluorescence microscopy. Agarose gel electrophoresis revealed that cells treated with piplartine exhibited DNA fragmentation. In addition, growth inhibition of PC-3 cells was associated with G2/M arrest and sub-G1 accumulation. Higher concentrations (24-30 microM) of piplartine modulated apoptosis-related protein expression by down-regulating cdc-2 expression and up-regulating PARP/procaspase-3 cleavage. Also, PC-3 cells treated with piplartine demonstrated caspase-3 activation, as observed with an in vitro caspase-3 colorimetric assay kit. Taken together, these results demonstrated that high concentrations of piplartine exhibited anti-proliferative and anti-cancer effects on PC-3 cells and that caspase-3-mediated PARP cleavage and cell cycle arrest at G2/M phase are involved in the underlying cellular mechanism of the apoptosis process.     

Statin induces apoptosis and cell growth arrest in prostate cancer cells.

Statins are a class of low molecular weight drugs that inhibit the rate-limiting enzyme of the mevalonate pathway 3-hydroxy-3-methylglutaryl-CoA reductase. Statins have been approved and effectively used to control hypercholesterolemia in clinical setting. Recent study showed statin's antitumor activity and suggested a potential role for prevention of human cancers. In this study, we did cell viability, DNA fragmentation, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays to evaluate the action of statins on prostate cancer cells and used Western blotting and RhoA activation assay to investigate the underlying molecular mechanism of action. Our data showed that lovastatin and simvastatin effectively decreased cell viability in three prostate cancer cell lines (PC3, DU145, and LnCap) by inducing apoptosis and cell growth arrest at G(1) phase. Both lovastatin and simvastatin induced activation of caspase-8, caspase-3, and, to a lesser extent, caspase-9. Both statins suppressed expression of Rb, phosphorylated Rb, cyclin D1, cyclin D3, CDK4, and CDK6, but induced p21 and p27 expression in prostate cancer cells. Furthermore, lovastatin and simvastatin suppressed RhoA activation and c-JUN expression, but not cyclooxygenase-2 expression. Our data showed that the antitumor activity of statins is due to induction of apoptosis and cell growth arrest. The underlying molecular mechanism of statin's action is mediated through inactivation of RhoA, which in turn induces caspase enzymatic activity and/or G(1) cell cycle. Future studies should focus on examining statins and other apoptosis-inducing drugs (e.g., cyclooxygenase-2 inhibitors or curcumin) together to assess their efficacy in prevention of prostate cancer.     

Adipocyte-fatty acid binding protein induces apoptosis in DU145 prostate cancer cells

Adipocyte-fatty acid binding protein (A-FABP) is a 14-15 kDa cytoplasmic protein that binds unesterified fatty acids (FA). It is believed that A-FABP is present in normal cells and disappears in cancer cells. Prostate cancer DU145 cells lack expression of A-FABP. Here, we report that transfection of A-FABP blocked growth of DU145 cells suggesting its role as a tumor suppressor. A-FABP transfected- prostate cancer DU145 cells underwent apoptosis when induced to overexpress A-FABP using an ecdysone-controlled expression system. DU145 cell cultures in complete medium exhibited a maximum of approximately 28% of apoptotic cells after 96 h of exposure to an ecdysone analog, Ponasterone A. We found that the possible mechanisms leading to the observed apoptotic effect may be due, in part, to an overexpression of tumor necrosis factor-alpha (TNF-alpha) and a moderate downregulation of transforming growth factor-alpha (TGF-alpha) in DU145 cells overexpressing A-FABP. The epidermal growth factor receptor (EGFR)/phosphatidyl inositol 3-kinase (PI 3-kinase) signaling pathway was not altered in these cells, suggesting that A-FABP may cause apoptosis by inducing downregulation of essential autocrine growth factors and/or upregulation of pro-apoptotic ones.     

DNA mismatch repair gene MLH1 induces apoptosis in prostate cancer cells

Mismatch repair (MMR) enzymes have been shown to be deficient in prostate cancer (PCa). MMR can influence the regulation of tumor development in various cancers but their role on PCa has not been investigated. The aim of the present study was to determine the functional effects of the mutL-homolog 1 (MLH1) gene on growth of PCa cells. The DU145 cell line has been established as MLH1-deficient and thus, this cell line was utilized to determine effects of MLH1 by gene expression. Lack of MLH1 protein expression was confirmed by Western blotting in DU145 cells whereas levels were high in normal PWR-1E and RWPE-1 prostatic cells. MLH1-expressing stable transfectant DU145 cells were then created to characterize the effects this MMR gene has on various growth properties. Expression of MLH1 resulted in decreased cell proliferation, migration and invasion properties. Lack of cell growth in vivo also indicated a tumor suppressive effect by MLH1. Interestingly, MLH1 caused an increase in apoptosis along with phosphorylated c-Abl, and treatment with MLH1 siRNAs countered this effect. Furthermore, inhibition of c-Abl with STI571 also abrogated the effect on apoptosis caused by MLH1. These results demonstrate MLH1 protects against PCa development by inducing c-Abl-mediated apoptosis.     

Apogossypolone induces autophagy and apoptosis in breast cancer MCF-7 cells in vitro and in vivo

Objective

Apogossypolone (ApoG2), a new derivative of gossypol, is a potent cell-growth inhibitor. ApoG2 has been demonstrated to have superior anti-tumor activity than gossypol in Bcl-2 transgenic mice. The purpose of this study was to investigate the inhibitory effect of ApoG2 on breast cancer cell line MCF-7 in vitro and in vivo, and to investigate its anti-tumor mechanism.

Methods

MCF-7 cell line in culture was treated with ApoG2. The inhibitory effects of ApoG2 on cell growth were measured by MTT and colony-formation assay. The cell apoptotic rate and cell cycle were analyzed by use of flow cytometry (FCM). The ultrastructural changes were observed by transmission electron microscopy. Autophagy was detected by acridine orange staining. Expression of Bcl-2, Bax, and Beclin 1 proteins was measured by western blot analysis.

Results

The inhibitory effect of ApoG2 on MCF-7 cell proliferation was dose and time-dependent. The maximum effect was observed when cells were incubated for 72 h with 40 μM ApoG2. ApoG2 at 5 μM also inhibited colony formation. FCM assay indicated that ApoG2 induced cell apoptosis and caused cell arrest in the S phase and G2/M phase. Transmission electron microscopic examination and acridine orange staining showed that ApoG2 induced intracellular autolysosome formation. Furthermore, ApoG2 reduced Bcl-2 expression, and enhanced expression of Bax and Beclin 1. Xenografting of MCF-7 cells in mice can also be inhibited by ApoG2.

Conclusion

ApoG2, a novel anti-apoptotic Bcl-2 agent, inhibits proliferation of breast cancer cell line MCF-7 by inducing cell apoptosis and autophagy.     

Ginsenoside F2 induces apoptosis accompanied by protective autophagy in breast cancer stem cells

Valproic acid (VPA) has extensive effects on leukemic blasts through its inhibition of histone deacetylases. The main goal of this study was to identify the subgroup of patients who may benefit most from VPA treatment. We examined the significance of t(8;21) chromosomal aberration for VPA treatment response among acute myeloid leukemia (AML) patients by direct comparison of AML1/ETO-negative vs. positive leukemic cell-lines as well as bone marrow blasts from AML patients. In t(8;21) AML, leukemogenesis is supposed to be induced via aberrant recruitment of histone deacetylases. AML cell lines of different genotypes (Kasumi-1, Kasumi-6, MV4;11, K562) and diagnostic bone marrow samples from patients were treated with VPA. VPA induced apoptosis in AML1/ETO-positive and MLL-AF4-positive cells in a dose-dependent manner. Differentiation, as indicated by changes in immunophenotype, was observed only in AML1/ETO-positive cells. VPA increased the expression of AML1 target genes - PU.1, C/EBPa, BPI and IGFBP7 only in AML1/ETO-positive cells. This AML1/ETO-specific effect was confirmed also using patient blasts isolated at the time of diagnosis. AML1/ETO-positive leukemia shows specific mechanism of VPA residing from differentiation followed by apoptosis that is accompanied by an increase in the expression of repressed AML1 target genes. Our data suggest that AML1/ETO-positive patients might derive the greatest benefit from VPA treatment.     

Ginsenoside F2 induces apoptosis accompanied by protective autophagy in breast cancer stem cells

Ginsenoside F2 (F2) was assessed for its antiproliferative activity against breast cancer stem cells (CSCs). F2 induced apoptosis in breast CSCs by activating the intrinsic apoptotic pathway and mitochondrial dysfunction. Concomitantly, F2 induced the formation of acidic vesicular organelles, recruitment of GFP-LC3-II to autophagosomes, and elevation of Atg-7 levels, suggesting that F2 initiates an autophagic progression in breast CSCs. Treatment with an inhibitor of autophagy enhanced F2-induced cell death. Our findings provide new insights into the anti-cancer activity of F2 and may contribute to the rational use and pharmacological study of F2.     

Synergy of Pancratistatin and Tamoxifen on breast cancer cells in inducing apoptosis by targeting mitochondria

Pancratistatin (PST), a natural compound obtained from the Hawaiian spider lily, is known to be specific and selective in inducing apoptosis in multiple cancer cell lines while sparing noncancerous cells and cell lines. Here we report the ability of PST to induce apoptosis specifically in human breast cancer cell lines MCF-7 and Hs-578-T compared to their non cancerous counterparts. In cancer cells PST caused increased levels of reactive oxygen species (ROS), decreased ATP and mitochondrial membrane permeabilization indicating the activation of the mitochondrial pathway of apoptosis. In combination with the anti-estrogen Tamoxifen, PST had a synergic effect. Both compounds caused increased production of ROS when applied to isolated mitochondria from these cancer cell lines supporting the observation that Tamoxifen might work through mechanisms distinct from the canonical estrogen receptor antagonism.     

Asparaginase induces apoptosis and cytoprotective autophagy in chronic myeloid leukemia cells

The antitumor enzyme asparaginase, which targets essential amino acid L-asparagine and catalyzes it to L-aspartic acid and ammonia, has been used for years in the treatment of acute lymphoblastic leukemia (ALL), subtypes of myeloid leukemia and T-cell lymphomas, whereas the anti-chronic myeloid leukemia (CML) effect of asparaginase and its underlying mechanism has not been completely elucidated. We have shown here that asparaginase induced significant growth inhibition and apoptosis in K562 and KU812 cells. Apart from induction of apoptosis, we reported for the first time that asparaginase induced autophagic response in K562 and KU812 cells as evidenced by the formation of autophagosome, microtubule-associated protein light chain 3 (LC3)-positive autophagy-like vacuoles, and the upregulation of LC3-II. Further study suggested that the Akt/mTOR (mammalian target of rapamycin) and Erk (extracellular signal-regulated kinase) signaling pathway were involved in asparaginase-induced autophagy in K562 cells. Moreover, blocking autophagy using pharmacological inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, chloroquine (CQ) and quinacrine (QN) enhanced asparaginase-induced cell death and apoptosis, indicating the cytoprotective role of autophagy in asparaginase-treated K562 and KU812 cells. Together, these findings provide a rationale that combination of asparaginase anticancer activity and autophagic inhibition might be a promising new therapeutic strategy for CML.     

Histone deacetylase inhibition induces apoptosis and autophagy in human neuroblastoma cells

Neuroblastoma (NB) is the most common solid extracranial tumor in children. Here we showed that trichostatin A, a histone deacetylase inhibitor (HDACi), decreases cell viability in three NB cell lines of different phenotypes. The treatment leads to G2/M-phase arrest, apoptosis and autophagy. Autophagy induction accompanies apoptosis in the most proliferative, N-Myc overexpressing cells. In contrast, autophagy precedes apoptosis and acts as a protective mechanism in the less proliferative, non-N-Myc overexpressing cells. Therefore, the autophagy induction is a relevant event in the NB response to HDACis, and it should be considered in the design of new treatments for this malignancy.