Differential anti-tumor activity of coriolus versicolor (Yunzhi) extract through p53- and/or Bcl-2-dependent apoptotic pathway in human breast cancer cells

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells, but the underlying mechanism has not been fully elucidated. The present study aimed to evaluate the in vitro anti-tumor activity of a standardized aqueous ethanol extract prepared from CV on four breast cancer cell lines using MTT assay, and test whether the mechanism involves apoptosis induction and modulation of p53 and Bcl-2 protein expressions using cell death detection ELISA, p53 and Bcl-2 ELISAs respectively. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of three breast tumor cell lines, with ascending order of IC50 values: T-47D, MCF-7, MDA-MB-231, while BT-20 cells were not significantly affected. Tumoricidal activity of the CV extract was found to be comparable to a chemotherapeutic anti-cancer drug, mitomycin C. Nucleosome productions in apoptotic MDA-MB-231, MCF-7 and T-47D cells were significantly augmented in a time-dependent manner and paralleled the anti-proliferative activity of CV extract. Expression of p53 protein was significantly upregulated only in T-47D cells treated with the CV extract in a dose- and time-dependent fashion, but not in MCF-7 (except at 400 mug/ml after 16 h) and MDA-MB-231 cells. The CV extract significantly induced a dose-dependent downregulation of Bcl-2 protein expression in MCF-7 and T-47D cells, but not in MDA-MB-231 cells. These results suggested that apoptosis induction, differentially dependent of p53 and Bcl-2 expressions, might be the possible mechanism of CV extract-mediated cytotoxicity in human breast cancer cells in vitro.     

丹参多酚酸盐通过线粒体途径诱导人肝癌SMMC-7721细胞的凋亡

目的：研究丹参多酚酸盐(salvianolate)体外诱导人肝癌细胞SMMC-7721凋亡作用及其可能机制。方法：不同质量浓度丹参多酚酸盐(0.5、1、2 mg/ml)与肝癌细胞共培养24 h后,流式细胞仪检测肝癌细胞凋亡,线粒体膜电位试剂盒(JC-1)检测线粒体膜电位变化;比色法测定1.0 mg/ml丹参多酚酸盐作用后肝癌细胞内caspase8、caspase9 及caspase3的活性,流式细胞仪检测培养体系内加入caspase9抑制剂(zLEHDfmk)或caspase3抑制剂(zDEVDfmk)后细胞凋亡率的变化,Western blotting检测肝癌细胞内线粒体凋亡途径相关蛋白Bax、Bcl2表达水平。结果：丹参多酚酸盐显著诱导肝癌细胞SMMC7721凋亡(P<0.05),同时线粒体膜电位随着药物浓度的升高而加剧下降(P<0.05)。1.0 mg/ml 丹参多酚酸盐处理肝癌细胞24 h后caspase-9与caspase-3的活性明显升高(P<0.05),而caspase-8的活性无明显变化(P>0.05);当培养体系内加入caspase-9或caspase3活性抑制剂后,丹参多酚酸盐诱导肿瘤细胞凋亡的作用明显降低(P<0.05)。Western blotting检测显示,丹参多酚酸盐处理组前凋亡蛋白Bax表达明显升高,抗凋亡蛋白Bcl2表达降低。结论：丹参多酚酸盐(0.5~2.0 mg/ml)剂量具有促进肝癌细胞凋亡的作用,且有剂量依赖的趋势,其机制与线粒体凋亡途径有关。     

Arsenic trioxide induces oxidative stress,DNA damage,and mitochondrial pathway of apoptosis in human leukemia (HL-60) cells

Background

Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML), which accounts for approximately 10% of all acute myloid leukemia cases. It is a blood cancer that is formed by chromosomal mutation. Each year in the United States, APL affects about 1,500 patients of all age groups and causes approximately 1.2% of cancer deaths. Arsenic trioxide (ATO) has been used successfully for treatment of APL patients, and both induction and consolidated therapy have resulted in complete remission. Recently published studies from our laboratory have demonstrated that ATO pharmacology as an anti-leukemic drug is associated with cytotoxic and genotoxic effects in leukemia cells.

Methods

In the present study, we further investigated the detailed molecular mechanism of ATO-mediated intrinsic pathway of apoptosis; using HL-60 cells as a test model. Oxidative stress was assessed by spectrophotometric measurements of MDA and GSH levels while genotoxicity was determined by single cell gel electrophoresis (Comet assay). Apoptosis pathway was analyzed by Western blot analysis of Bax, Bcl2 and caspase 3 expression, as well as immunocytochemistry and confocal imaging of Bax and Cyt c translocation and mitochondrial membrane potential depolarization.

Results

ATO significantly (p < 0.05) induces oxidative stress, DNA damage, and caspase 3 activityin HL-60 cells in a dose-dependent manner. It also activated the intrinsic pathway of apoptosis by significantly modulating (p < 0.05) the expression and translocation of apoptotic molecules and decreasing the mitochondrial membrane potential in leukemia cells.

Conclusion

Taken together, our research demonstrated that ATO induces mitochondrial pathway of apoptosis in HL-60 cells. This apoptotic signaling is modulated via oxidative stress, DNA damage, and change in mitochondrial membrane potential, translocation and upregulation of apoptotic proteins leading programmed cell death.     

Corosolic acid induces apoptosis through mitochondrial pathway and caspases activation in human cervix adenocarcinoma HeLa cells

We investigated the response of human cervix adenocarcinoma HeLa cells to Corosolic acid (CRA) treatment. Our results showed that CRA significantly inhibited cell viability in both a dose- and a time-dependent manner. CRA treatment induced S cell-cycle arrest and caused apoptotic death in HeLa cells. We found that CRA increased in Bax/Bcl-2 ratios by up-regulating Bax expression, disrupted mitochondrial membrane potential and triggered the release of cytochrome c from mitochondria into the cytoplasm. Moreover, CRA treatment triggered the activation of caspase-8, -9 and -3 in HeLa cells. All these results indicate that CRA-induced apoptosis is associated with the activation of caspases via a mitochondrial pathway. Taken together, we believe that CRA could have strong potentials for clinical application in treating human cervix adenocarcinoma and improving cancer chemotherapy.     

Hyperthermic CO2 pneumoperitoneum induces apoptosis in human colon cancer cells through Bax-associated mitochondrial pathway

Peritoneal carcinomatosis of colorectal cancer is common and associated with poor prognosis, which poses a serious challenge and satisfactory treatments are urgently needed. Hyperthermic CO2 pneumoperitoneum (HT-CO2) is a new strategy. This study was designed to determine the potential of HT-CO2 against colorectal cancer cells. Based on an in vitro HT-CO2 study model, the anti-tumor efficacy of HT-CO2 (42-44 degrees C for 2-4 h) on human colon cancer COLO 205 cells was evaluated and the mechanisms of actions were analyzed. We found that HT-CO2 (43-44 degrees C for 2-4 h) significantly decreased cell viability as determined by WST-8 assay, and the cytotoxicity was attributable to HT-CO2-induced hyperthermia and extracellular acidification. Apoptosis was the major form of cell killing as demonstrated by Annexin-V/PI flow cytometry and morphological analysis (Hoechst/PI fluorescence microscopy and transmission electron microscopy). Further Western blot analysis and flow cytometric analysis of mitochondrial membrane potential showed that Bax-associated mitochondrial apoptotic pathway played critical role in the induction of apoptosis. We conclude that HT-CO2 has significant cytotoxic effect on colon cancer cells through induction of Bax-associated mitochondrial apoptosis, and the cytocidal effect is attributable to HT-CO2-induced hyperthermia and extracellular acidifications. Our data suggest that HT-CO2 may serve as a potential candidate for treating and/or preventing peritoneal carcinomatosis of colorectal cancer and further investigations both in vitro as well as in vivo in animal models are needed.     

Physalis peruviana extract induces apoptosis in human Hep G2 cells through CD95/CD95L system and the mitochondrial signaling transduction pathway

Physalis species is a popular folk medicine used for treating cancer, leukemia, hepatitis and other diseases. Studies have shown that the ethanol extract of Physalis peruviana (EEPP) inhibits growth and induces apoptotic death of human Hep G2 cells in culture, whereas proliferation of the mouse BALB/C normal liver cells was not affected. In this study, we performed detailed studies to define the molecular mechanism of EEPP-induced apoptosis in Hep G2 cells. The results further confirmed that EEPP inhibited cell proliferation in a dose- and time-dependent manner. At 50 microg/ml, EEPP significantly increased the accumulation of the sub-G1 peak (hypoploid) and the portion of apoptotic annexin V positive cells. EEPP was found to trigger apoptosis through the release of cytochrome c, Smac/DIABLO and Omi/HtrA2 from mitochondria to cytosol and consequently resulted in caspase-3 activation. Pre-treatment with a general caspase inhibitor (z-VAD-fmk) prevented cytochrome c release. After 48 h of EEPP treatment, the apoptosis of Hep G2 cells was found to associate with an elevated p53, and CD95 and CD95L proteins expression. Furthermore, a marked down-regulation of the expression of the Bcl-2, Bcl-XL and XIAP, and up-regulation of the Bax and Bad proteins were noted. Taken together, the present results suggest that EEPP-induced Hep G2 cell apoptosis was possibly mediated through the CD95/CD95L system and the mitochondrial signaling transduction pathway.     

VI-16, a newly synthesized flavonoid, induces apoptosis through the mitochondrial pathway in human hepatoma cells

VI-16, a newly synthesized flavonoid, has a hydroxy substitution at C5 position, a methoxyl substitution at C5 position, and a piperazine substitution at C7 position. Here, we firstly investigated the potential antitumor effect of VI-16 in HepG2 human hepatocarcinoma cells. The MTT assay showed that VI-16 inhibited HepG2 cell growth in a concentration- and time-dependent manner. To further investigate whether apoptosis induction contributed to the antitumor effects of VI-16, DAPI staining and Annexin-V/PI double staining were performed in our tests. The data showed that VI-16 could induce apoptotic cell death in HepG2 cells. Moreover, mechanistic studies revealed that VI-16-induced apoptosis was a caspase-dependent process by decreasing the expression of pro-caspase-3. The changes in the expression of caspase-8, capsase-9, Bax and bcl-2 after VI-16 treatment suggested that the mitochondrial pathway was involved in the apoptosis induced by VI-16. Furthermore, VI-16 could significantly increase the loss of mitochondrial membrane potential and the expression of p53. Taken together, these results demonstrated that apoptosis induced by VI-16 might be one of the mechanisms by which VI-16 acts as a preventive antitumor drug against human hepatoma.     

Embelin induces apoptosis through down-regulation of XIAP in human leukemia cells

The present study was undertaken to determine the molecular mechanisms by which embelin induces apoptosis in human leukemia cells. Embelin resulted in loss of cell viability and inhibition of proliferation in a dose- and time-dependent manner, which was largely attributed to apoptosis. Embelin caused depolarization of mitochondrial membrane potential. Western blot analysis showed that the expression of anti-apoptotic proteins X-linked inhibitor of apoptosis (XIAP) was down-regulated by embelin. Embelin induced activation of caspase-9 and embelin-induced apoptosis was prevented by caspase inhibitors. Taken together, these findings suggest that embelin results in human leukemia cells apoptosis through caspase-dependent mechanisms involving down-regulation of XIAP.     

Ethanolic extract of fermented Thunb induces human leukemic HL-60 and Molt-4 cell apoptosis via oxidative stress and a mitochondrial pathway

Houttuynia cordata Thunb (HCT) is a medicinal plant of the Saururaceae family which features antimutagenic and antiviral properties. For extraction, the whole plants were fermented or non-fermented with yeast and ethanol then the whole plants were dried, ground and extracted with 95% ethanol or water. The aims of this study were to compare cytotoxic effects, apoptosis induction, and mechanism(s) with the ethanolic and water extracts of fermented and non-fermented HCT. Cytotoxicity was assessed using the MTT assay in human leukemic HL-60, Molt-4 and peripheral blood mononuclear cells (PBMCs). Apoptotic death was characterized by staining with propidium iodide and examined under a fluorescence microscope. Peroxide radical production and reduction of mitochondrial transmembrane potential (MTP) were determined using 2',7'-dichlorohydrofluorescein diacetate and 3,3'-dihexyloxacarbocyanine iodide and flow cytometry, respectively. The expression of caspase-9 was identified by immunoblotting. The ethanolic extract of fermented HCT was cytotoxic to HL-60 >Molt- 4 > PBMCs, to a greater extent than the non-fermented preparation and the number of apoptotic cells was higher. The alcoholic (fermented) extract produced more radicals than the non-fermented in HL-60 cells but the converse was observed in Molt-4 cells. Reduction of MTP was found in HL-60 and Molt-4 cells treated with the alcoholic (fermented) extract and caspase-9 was cleaved dose-dependently in both cells. In conclusion, the alcoholic extract of fermented HCT was more toxic to human leukemic cells than the non-fermented and both cell lines underwent apoptosis via oxidative stress and a mitochondrial pathway.     

Ceramide induces mitochondrial activation and apoptosis via a Bax-dependent pathway in human carcinoma cells

The intracellular pathways leading to mitochondrial activation and subsequent cell death in the ceramide-mediated stress response have been intensively studied in recent years. Experimental evidence has been provided that ceramide-induced apoptosis is inhibited by overexpression of antiapoptotic proteins of the Bcl-2 family. However, the direct effect of proapoptotic gene products, e.g. Bax, on ceramide-induced death signalling has not yet been studied in detail. In the present work, we show by measurement of mitochondrial permeability transition, cytochrome c release, activation of caspase-3 and DNA fragmentation that ceramide-induced apoptosis is marginal in Bax-negative DU 145 cells. Reconstitution of Bax by generation of DU 145 cells stably expressing this proapoptotic factor, clearly enhanced ceramide-induced apoptosis at all levels of the mitochondrial signalling cascade. Using the broad-range caspase inhibitor zVAD-fmk and zDEVD-fmk, an inhibitor of caspase-3-like activities, we demonstrate that the ceramide-induced mitochondrial activation in Bax-transfected DU 145 cells is caspase-independent. On the other hand, apoptotic events located downstream of the mitochondria, e.g. DNA fragmentation, were shown to be caspase-dependent. This influence of Bax on ceramide-induced apoptosis was confirmed in another cellular system: whereas Bax-positive HCT116 wild type cells were very sensitive towards induction of cell death by C(2)-ceramide, sensitivity of Bax knock-out HCT116 cells was significantly reduced. Thus, we conclude that Bax is a key activator of ceramide-mediated death pathways.     

The protein kinase Cbeta-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses signaling through the AKT pathway, induces apoptosis, and suppresses growth of human colon cancer and glioblastoma xenografts

Activation of protein kinase Cbeta (PKCbeta) has been repeatedly implicated in tumor-induced angiogenesis. The PKCbeta-selective inhibitor, Enzastaurin (LY317615.HCl), suppresses angiogenesis and was advanced for clinical development based upon this antiangiogenic activity. Activation of PKCbeta has now also been implicated in tumor cell proliferation, apoptosis, and tumor invasiveness. Herein, we show that Enzastaurin has a direct effect on human tumor cells, inducing apoptosis and suppressing the proliferation of cultured tumor cells. Enzastaurin treatment also suppresses the phosphorylation of GSK3betaser9, ribosomal protein S6(S240/244), and AKT(Thr308). Oral dosing with Enzastaurin to yield plasma concentrations similar to those achieved in clinical trials significantly suppresses the growth of human glioblastoma and colon carcinoma xenografts. As in cultured tumor cells, Enzastaurin treatment suppresses the phosphorylation of GSK3beta in these xenograft tumor tissues. Enzastaurin treatment also suppresses GSK3beta phosphorylation to a similar extent in peripheral blood mononuclear cells (PBMCs) from these treated mice. These data show that Enzastaurin has a direct antitumor effect and that Enzastaurin treatment suppresses GSK3beta phosphorylation in both tumor tissue and in PBMCs, suggesting that GSK3beta phosphorylation may serve as a reliable pharmacodynamic marker for Enzastaurin activity. With previously published reports, these data support the notion that Enzastaurin suppresses tumor growth through multiple mechanisms: direct suppression of tumor cell proliferation and the induction of tumor cell death coupled to the indirect effect of suppressing tumor-induced angiogenesis.     

Allicin inhibits cell growth and induces apoptosis in U87MG human glioblastoma cells through an ERK-dependent pathway

Allicin, the main flavor compound in garlic, has anti-carcinogenic activities in a range of cancer cells, however, the underlying molecular mechanisms are not completely understood. This study examined the effect of allicin on the cell viability of U87MG human glioma cells along with its molecular mechanisms of induction of cell death. Apoptosis was determined by TUNEL and Hoechst 33258 staining as well as by western blot analysis. Allicin inhibited the cell viability of U87MG human glioma cells in a dose- and time-dependent manner. Allicin-induced inhibition of cell viability was due to apoptosis of cells. The mechanisms of apoptosis were found to involve the mitochondrial pathway of Bcl-2/Bax, the MAPK/ERK signaling pathway and antioxidant enzyme systems. These results suggest that allicin can serve as a novel chemotherapeutic candidate for the treatment of glioblastoma multiforme.     

Nitric oxide induces promyelocytic cell growth arrest and apoptosis through deactivation of Akt pathway

It is NO that mediates the inhibitory effect of IFN-gamma, MIP-1alpha and TNFalpha on hematopoiesis. However, the mechanism for NO effect on the hematopoietic system is likely to not clear. In the current work, we demonstrates that NO can directly suppress the colony formation of granulocyte/macrophage in vitro. Using a granulocyte/macrophage progenitor (GMP) model, HL-60 cell line, we show that NO inhibits the proliferation of HL-60 cells by inducing G0/G1 arrest and apoptosis in a dose- and time-dependent manner. Exposure of HL-60 cells to 1mM SNP for 2-48h results in marked decrease in Akt activation and Bad phosphorylation. Constitutively active Akt overexpression reduces NO-induced apoptosis and cell cycle arrest in HL-60 cells. A further investigation on apoptosis related protein shows that NO induces Bid cleavage and Bax expression but down-regulates the expression of Bcl-2 and Bcl-xL. We also demonstrate here that G0/G1 arrest is resulted from NO-induced disruption of cell cycle balance, which is mediated by up-regulation of p21(waf/cip1), p27(kip1) and down-regulation of cyclin D1, cyclin E. In brief, NO-induced apoptosis and G0/G1 arrest is mediated through regulation of apoptosis and cell cycle related protein, which may depend on Akt deactivation by NO, ultimately led to its inhibitory effect on hematopoiesis.     

N-(beta-Elemene-13-yl)tryptophan methyl ester induces apoptosis in human leukemia cells and synergizes with arsenic trioxide through a hydrogen peroxide dependent pathway

beta-Elemene is an active component of herb medicine Curcuma Wenyujin and N-(beta-elemene-13-yl)tryptophan methyl ester (ETME) was synthesized for increasing its antitumor activity. ETME induced apoptosis in human leukemia HL-60 and NB4 cells at concentrations less than 40muM. The apoptosis induction ability of ETME was associated with the production of hydrogen peroxide (H(2)O(2)), the decrease of mitochondrial membrane potential, and the activation of caspase-3 that was blocked by catalase. ETME in combination with arsenic trioxide (As(2)O(3)), an agent used to treat acute promyelocytic leukemia, synergistically induced apoptosis in both cell lines by enhanced production of H(2)O(2). These data suggest that ETME induces apoptosis and synergizes with As(2)O(3) in leukemia cells through a H(2)O(2)-dependent pathway.     

Rapamycin induces p53-independent apoptosis through the mitochondrial pathway in non-small cell lung cancer cells

The mammalian target of rapamycin (mTOR) is a key kinase acting downstream of growth factor receptor PI3K and AKT signaling, leading to processes resulting in increased cell size and proliferation through translation control. Rapamycin, a specific inhibitor of mTOR, results predominately in G1 cell cycle arrest through translation control and occasionally, cell type-dependent apoptosis by an unknown mechanism. In this study, we investigated the effect and mechanism of action of rapamycin on non-small cell lung cancer (NSCLC) cell lines with p53 mutations. Cell proliferation was evaluated by modified MTT assay. The apoptotic effect of rapamycin was measured by caspase-3 activation and flow cytometric analysis of Annexin V binding. The expression of Bcl-2 and the release of cytochrome?c from mitochondria were evaluated by western blotting. We found that rapamycin induced apoptosis in NSCLC cell lines with p53 mutations. Western blot analysis demonstrated that rapamycin downregulates the expression levels of Bcl-2, which leads to increased cytochrome c release from mitochondria and subsequent activation of caspase cascades. These findings suggest that rapamycin induces p53-independent apoptosis through downregulation of Bcl-2 and the mitochondrial pathway in NSCLC cell lines as a novel antitumor mechanism.     

Curcumol induces apoptosis via caspases-independent mitochondrial pathway in human lung adenocarcinoma ASTC-a-1 cells

Curcumol isolated from Rhizoma Curcumae, a common traditional Chinese medicine, has been reported to possess antitumor effects, but little is known about the molecular mechanism. The current study tried to explore the molecular mechanism of curcumol-induced cell death in human lung adenocarcinoma (ASTC-a-1) cells. Cell counting kit (CCK-8) assay, fluorescence resonance energy transfer (FRET), confocal fluorescence microscopy imaging, emission spectral analysis, and flow cytometry (FCM) technologies were used to assess the characteristics of curcumol-induced cell death. FCM analysis showed that curcumol induced G₂/M phase arrest, nuclear fragmentation and phosphatidylserine externalization, implying that curcumol dominantly induced apoptotic cell death. Dynamical imaging showed that curcumol induced a rapid translocation of Bax from cytosol into mitochondria within 6 h, leading to a rapid dissipation of mitochondrial membrane potential (∆Ψm), implying that mitochondria play an important role in the curcumol-induced apoptosis. The fact that Z-VAD-fmk, a broad-spectrum inhibitor of caspases, did not reduce the curcumol-induced apoptosis showed that caspases were not involved in curcumol-induced apoptosis, which was further verified by monitoring the caspase-3 activation inside living cells stably expressing SCAT3, a FRET plasmid. Collectively, we for the first time demonstrated that curcumol induced cell death in a dominant apoptotic fashion via the caspases-independent mitochondrial pathway in ASTC-a-1 cells.     

The chemopreventive agent N-(4-hydroxyphenyl)retinamide induces apoptosis through a mitochondrial pathway regulated by proteins from the Bcl-2 family

N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a potent chemopreventive agent whose effect has been suggested to involve apoptosis induction. 4-HPR induces a loss of the mitochondrial transmembrane potential and the mitochondrial release of cytochrome c before caspase activation. Inhibition of mitochondrial membrane permeabilization (MMP) by transfection with Bcl-2 or the Cytomegalovirus UL37 gene product vMIA prevented caspase activation and cell death. In contrast to other retinoid derivatives, 4-HPR has no direct MMP-inducing effects when added to isolated mitochondria or when added to proteoliposomes containing the MMP-regulatory permeability transition pore complex (PTPC). Moreover, although reactive oxygen species (ROS) overproduction appears to be instrumental for 4-HPR-induced MMP and apoptosis, inhibition of the NF-kappaB or p53-mediated signal transduction pathways failed to modulate 4-HPR-induced apoptosis. 4-HPR was found to cause an antioxidant-inhibitable conformational change of both Bax and Bak, leading to the exposure of their N-termini and to the mitochondrial relocalization of Bax. Cells with a Bax(-/-) Bak(-/-) genotype were resistant against the 4-HPR-induced MMP, overproduction of ROS and cell death. Altogether, these data indicate that 4-HPR induces MMP through an ROS-mediated pathway that involves the obligatory contribution of the proapopotic Bcl-2 family members Bax and/or Bak.