Synthetic retinoid CD437 induces mitochondria-mediated apoptosis in hepatocellular carcinoma cells

Retinoids play an important role in the regulation of cell growth and death. Synthetic retinoid CD437 reportedly induces apoptosis in various cancer cell lines. However, the mechanism of inducing apoptosis in hepatocellular carcinoma (HCC) cells by this agent remains to be clarified. In this study, we investigated the signaling pathway by which CD437 induces apoptosis in HCC cell lines. Apoptosis of six human HCC cell lines was induced by treatment with CD437. Caspase-3 and -9 were activated by CD437, suggesting that the apoptosis is mediated by mitochondrial pathways. Consistent with these findings, the treatment with CD437 upregulated Bax protein, downregulated Bcl-2 protein and released cytochrome c into the cytoplasm. Moreover, rhodamine123 staining revealed mitochondrial depolarization in the cells treated with CD437. These data of the present study suggest that CD437 induces apoptosis in HCC cells via mitochondrial pathways.     

Synthetic retinoid CD437 induces apoptosis and acts synergistically with TRAIL receptor-2 agonist in malignant melanoma

The novel synthetic retinoid, CD437, shows potent anti-tumor activity in a range of different cancer cell lines and now serves as a prototype for development of new retinoid related molecules (RRMs). The purpose of this study was to examine the effect and cellular targets of CD437 in the human metastatic melanoma cell lines FEMX-1 and WM239. We showed that treatment with CD437 led to cell cycle arrest and induced apoptosis through both the extrinsic- and intrinsic pathways (caspase 8, -9 and PARP cleavage) in both cell lines. Interestingly, apoptosis was induced independently of DNA-fragmentation in FEMX-1 cells, and appeared partially caspase-independent in the WM239 cells. Additionally, up-regulation of CHOP mRNA and cathepsin D protein expression, following retinoid treatment, suggests involvement of the endoplasmatic reticulum (ER) and lysosomes, respectively. Combination of suboptimal concentrations of CD437 and lexatumumab, a TRAIL death receptor-2 agonist, resulted in synergistic reduction of viable cells, along with increased PARP cleavage. These results indicate that CD437 has a strong anti-neoplastic effect alone and in combination with lexatumumab in melanoma cell lines.     

CD437, a synthetic retinoid, induces apoptosis in human respiratory epithelial cells via caspase-independent mitochondrial and caspase-8-dependent pathways both up-regulated by JNK signaling pathway

The synthetic retinoid-related molecule CD437-induced apoptosis in human epithelial airway respiratory cells: the 16HBE bronchial cell line and normal nasal epithelial cells. CD437 caused apoptosis in S-phase cells and cell cycle arrest in S phase. Apoptosis was abolished by caspase-8 inhibitor z-IETD-fmk which preserved S-phase cells but was weakly inhibited by others selective caspase-inhibitors, indicating that caspase-8 activation was involved. z-VAD and z-IETD prevented the nuclear envelope fragmentation but did not block the chromatin condensation. The disruption of mitochondrial transmembrane potential was also induced by CD437 treatment. The translocation of Bax to mitochondria was demonstrated, as well as the release of cytochrome c into the cytosol and of apoptosis-inducing factor (AIF) translocated into the nucleus. z-VAD and z-IETD did not inhibit mitochondrial depolarization, Bax translocation or release of cytochrome c and AIF from mitochondria. These results suggest that CD437-induced apoptosis is executed by two converging pathways. AIF release is responsible for chromatin condensation, the first stage of apoptotic cell, via a mitochondrial pathway independent of caspase. But final stage of apoptosis requires the caspase-8-dependent nuclear envelope fragmentation. In addition, using SP600125, JNK inhibitor, we demonstrated that CD437 activates the JNK-MAP kinase signaling pathway upstream to mitochondrial and caspase-8 pathways. Conversely, JNK pathway inhibition, which suppresses S-phase apoptosis, did not prevent cell cycle arrest within S phase, confirming that these processes are triggered by distinct mechanisms.     

CD437 induces apoptosis in ovarian adenocarcinoma cells via ER stress signaling

A synthetic retinoid, CD437, has been shown to exert potent anti-tumor activity against various types of cancer cell lines, regardless of their sensitivities to natural retinoids. We herein demonstrate that CD437 induces endoplasmic reticulum (ER) stress, including the up-regulation of CHOP, BIP and GADD34 mRNA through ER stress transducer (PERK and IRE1α) activation in an ovarian adenocarcinoma cell line, SKOV3. It was also shown that CD437 induced the CHOP and GADD34 expressions in another four ovarian adenocarcinoma cell lines, indicating that CD437 functions as an ER stress inducer in these cell lines. Moreover, the siRNA-mediated knockdown of inducible CHOP expression prevented the cytotoxic effect of CD437. These results suggest that ER stress plays an important role in the mechanism by which CD437 induces apoptosis in ovarian adenocarcinoma cells.     

Evidence of a lysosomal pathway for apoptosis induced by the synthetic retinoid CD437 in human leukemia HL-60 cells.

The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene carboxylic acid (AHPN/CD437) has been proven to be a potent inducer of apoptosis in a variety of tumor cell types. However, the mechanism of its action remains to be elucidated. Recent studies suggest that the lysosomal protease cathepsin D, when released from lysosomes to the cytosol, can initiate apoptosis. In this study, we examined whether cathepsin D and free radicals are involved in the CD437-induced apoptosis. Exposure of human leukemia HL-60 cells to CD437 resulted in rapid induction of apoptosis as indicated by caspase activation, phosphatidylserine exposure, mitochondrial alterations and morphological changes. Addition of the antioxidants alpha-tocopherol acetate effectively inhibited the CD437-induced apoptosis. Measurement of the intracellular free radicals indicated a rise in oxidative stress in CD437-treated cells, which could be attenuated by alpha-tocopherol acetate. Interestingly, pretreatment of cells with the cathepsin D inhibitor pepstatin A blocked the CD437-induced free radical formation and apoptotic effects, suggesting the involvement of cathepsin D. However, Western blotting revealed no difference in cellular quantity of any forms of cathepsin D between control cells and CD437-treated cells, whereas immunofluorescence analysis of the intracellular distribution of cathepsin D showed release of the enzyme from lysosomes to the cytosol. Labeling of lysosomes with lysosomotropic probes confirmed that CD437 could induce lysosomal leakage. The CD437-induced relocation of cathepsin D could not be prevented by alpha-tocopherol acetate, suggesting that the lysosomal leakage precedes free radical formation. Furthermore, a retinoic acid nuclear receptor (RAR) antagonist failed to block these effects of CD437, suggesting that the action of CD437 is RAR-independent. Taken together, these data suggest a novel lysosomal pathway for CD437-induced apoptosis, in which lysosomes are the primary target and cathepsin D and free radicals act as death mediators.     

Nitric oxide induces apoptosis via AP-1-driven upregulation of COX-2 in rat pheochromocytoma cells

Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin synthesis, is induced in many cells by numerous inflammatory mediators, including nitric oxide (NO). Upregulation of COX-2 expression has been implicated in the pathophysiology of neuronal cell death. In the present study, we have found that the NO-induced upregulation of COX-2 via activation of activator protein-1 (AP-1) signaling leads to apoptotic cell death. Cultured rat pheochromocytoma (PC12) cells treated with sodium nitroprusside (SNP), a NO-releasing compound, exhibited marked induction of COX-2 expression, which was associated with apoptotic cell death as evidenced by internucleosomal DNA fragmentation, cleavage of poly(ADP-ribose) polymerase, activation of caspase-3, accumulation of p53, increased Bax/Bcl-XL ratio, and dissipation of mitochondrial membrane potential. In addition to the upregulation of COX-2 expression, SNP treatment led to activation of AP-1. Pretreatment of PC12 cells with c-fos antisense oligonucleotide abolished the NO-induced increase in DNA binding of AP-1 and upregulation of COX-2 expression. Furthermore, pretreatment with a selective COX-2 inhibitor (SC58635) rescued the PC12 cells from the apoptotic cell death induced by NO. Similar results were obtained when the NO-induced upregulation of COX-2 expression was blocked by the siRNA interference. These results suggest that excessive NO production during inflammation induces apoptosis in PC12 cells through AP-1-mediated upregulation of COX-2 expression.     

Cassane diterpenoid derivative induces apoptosis in IDH1 mutant glioma cells through the inhibition of glutaminase in vitro and in vivo

BackgroundGlioblastoma multiforme (GBM) is the most frequent, lethal and aggressive tumour of the central nervous system in adults. The discovery of novel anti-GBM agents based on the isocitrate dehydrogenase (IDH) mutant phenotypes and classifications have attracted comprehensive attention.PurposeDiterpenoids are a class of naturally occurring 20-carbon isoprenoid compounds, and have previously been shown to possess high cytotoxicity for a variety of human tumours in many scientific reports. In the present study, 31 cassane diterpenoids of four types, namely, butanolide lactone cassane diterpenoids (I) (1-10), tricyclic cassane diterpenoids (II) (11-15), polyoxybutanolide lactone cassane diterpenoids (III) (16-23), and fused furan ring cassane diterpenoids (IV) (24-31), were tested for their anti-glioblastoma activity and mechanism underlying based on IDH1 mutant phenotypes of primary GBM cell cultures and human oligodendroglioma (HOG) cell lines.ResultsWe confirmed that tricyclic-type (II) and compound 13 (Caesalpin A, CSA) showed the best anti-neoplastic potencies in IDH1 mutant glioma cells compared with the other types and compounds. Furthermore, the structure-relationship analysis indicated that the carbonyl group at C-12 and an α, β-unsaturated ketone unit fundamentally contributed to enhancing the anti-glioma activity. Studies investigating the mechanism demonstrated that CSA induced oxidative stress via causing glutathione reduction and NOS activation by negatively regulating glutaminase (GLS), which proved to be highly dependent on IDH mutant type glioblastoma. Finally, GLS overexpression reversed the CSA-induced anti-glioma effects in vitro and in vivo, which indicated that the reduction of GLS contributed to the CSA-induced proliferation inhibition and apoptosis in HOG-IDH1-mu cells.ConclusionTherefore, the present results demonstrated that compared with other diterpenoids, tricyclic-type diterpenoids could be a targeted drug candidate for the treatment of secondary IDH1 mutant type glioblastoma through negatively regulating GLS.     

Conditional expression of exogenous Bcl-X(S) triggers apoptosis in human melanoma cells in vitro and delays growth of melanoma xenografts

The Bcl-2-related proteins Bcl-X(L) and Bcl-X(S) represent alternative splice products and exert opposite activities in the control of apoptosis, but their significance for melanoma is not yet clear. Applying the tetracycline-inducible expression system Tet-On, we found overexpression of Bcl-X(S) by itself sufficient to induce apoptosis in vitro in stably transfected human melanoma cell lines. Combination with proapoptotic agents such as etoposide, pamidronate, and ceramide resulted in additive proapoptotic effects, whereas Bcl-X(L) protected from apoptosis caused via CD95/Fas stimulation. In nude mice growth of melanoma xenotransplants derived from stably transfected cells was significantly reduced after induction of Bcl-X(S) by doxycycline. Our results indicate that Bcl-X proteins are of major importance for control of apoptosis in malignant melanoma.     

Pharmacological inhibition of USP7 suppresses growth and metastasis of melanoma cells in vitro and in vivo

Melanoma is a highly aggressive type of skin cancer. The development of diverse resistance mechanisms and severe adverse effects significantly limit the efficiency of current therapeutic approaches. Identification of the new therapeutic targets involved in the pathogenesis will benefit the development of novel therapeutic strategies. The deubiquitinase ubiquitin–specific protease-7, a potential target for cancer treatment, is deregulated in types of cancer, but its role in melanoma is still unclear. We investigated the role and the inhibitor P22077 of ubiquitin-specific protease-7 in melanoma treatment. We found that ubiquitin-specific protease-7 was overexpressed and correlated with poor prognosis in melanoma. Further, pharmacological inhibition of ubiquitin-specific protease-7 by P22077 can effectively inhibit proliferation, and induce cell cycle arrest and apoptosis via ROS accumulation–induced DNA damage in melanoma cells. Inhibition of ubiquitin-specific protease-7 by P22077 also inhibits melanoma tumour growth in vivo. Moreover, inhibition of ubiquitin-specific protease-7 prevented migration and invasion of melanoma cells in vitro and in vivo by decreasing the Wnt/β-catenin signalling pathway. Taken together, our study revealed that ubiquitin-specific protease-7 acted as an oncogene involved in melanoma cell proliferation and metastasis. Therefore, ubiquitin-specific protease-7 may serve as potential candidates for the treatment of melanoma.     

Icaritin causes sustained ERK1/2 activation and induces apoptosis in human endometrial cancer cells

Icaritin, a compound from Epimedium Genus, has selective estrogen receptor (ER) modulating activities, and possess anti-tumor activity. Here, we examined icaritin effect on cell growth of human endometrial cancer Hec1A cells and found that icaritin potently inhibited proliferation of Hec1A cells. Icaritin-inhibited cell growth was associated with increased levels of p21 and p27 expression and reduced cyclinD1 and cdk 4 expression. Icaritin also induced cell apoptosis accompanied by activation of caspases as evidenced by the cleavage of endogenous substrate Poly (ADP-ribose) polymerase (PARP) and cytochrome c release, which was abrogated by pretreatment with the pan-caspase inhibitor z-VAD-fmk. Icaritin treatment also induced expression of pro-apoptotic protein Bax with a concomitant decrease of Bcl-2 expression. Furthermore, icaritin induced sustained phosphorylation of extracellular signal-regulated kinase1/2 (the MAPK/ ERK1/2) in Hec1A cells and U0126, a specific MAP kinase kinase (MEK1/2) inhibitor, blocked the ERK1/2 activation by icaritin and abolished the icaritin-induced growth inhibition and apoptosis. Our results demonstrated that icaritin induced sustained ERK 1/2 activation and inhibited growth of endometrial cancer Hec1A cells, and provided a rational for preclinical and clinical evaluation of icaritin for endometrial cancer therapy.     

Ginkgetin induces apoptosis via activation of caspase and inhibition of survival genes in PC-3 prostate cancer cells

Ginkgetin is a natural biflavonoid isolated from leaves of Ginkgo biloba L. Though it was known to have anti-inflammatory, anti-influenza virus, anti-fungal activity, osteoblast differentiation stimulating activity and neuro-protective effects, the underlying antitumor mechanism of ginkgetin still remains unclear. Thus, in the present study, anti-cancer mechanism of ginkgetin was elucidated in human prostate cancer PC-3 cells. Ginkgetin suppressed the viability of PC-3 cells in a concentration-dependent manner and also significantly increased the sub-G1 DNA contents of cell cycle in PC-3 cells. Ginkgetin activated caspase-3 and attenuated the expression of survival genes such as Bcl-2, Bcl-x_L, survivin and Cyclin D1 at protein and mRNA levels. Consistently, pan-caspase inhibitor Z-DEVD-fmk blocked sub G1 accumulation and cleavages of PRAP and caspase 3 induced by ginkgetin in PC-3 cells. Overall, these findings suggest that ginkgetin induces apoptosis in PC-3 cells via activation of caspase 3 and inhibition of survival genes as a potent chemotherapeutic agent for prostate cancer treatment.     

Ad-IL-24对MG-63人骨肉瘤细胞抑癌效应的体内外实验

本研究旨在分析腺病毒携带的IL-24基因在体内外对人骨肉瘤细胞生长抑制效应及其分子机制。将Ad-IL-24重组腺病毒感染MG-63细胞,用荧光显微镜、RT-PCR法检测IL-24在MG-63细胞中的转录和表达;MTT法、流式细胞技术和Hoechst染色法检测IL-24基因的表达对MG-63细胞的生长抑制和凋亡效应;半定量RT-PCR法检测IL-24基因的表达对MG-63细胞中的bcl-2、bax、caspase-3相关基因表达的影响。用Ad-IL-24重组腺病毒在MG-63骨肉瘤荷瘤裸鼠的瘤体内进行注射治疗,观察肿瘤生长变化,15d后处死裸鼠,摘除瘤体,称瘤重。并通过免疫组化法检测Bcl-2、Bax、Caspase-3等与细胞凋亡相关因子的表达。结果表明Ad-IL-24重组腺病毒感染MG-63细胞后,能明显抑制MG-63细胞增殖,并能通过上调细胞中bax、caspase-3和下调bcl-2基因表达,诱导细胞凋亡,呈现出典型细胞凋亡形态学变化。Ad-IL-24重组腺病毒瘤内注射MG-63裸鼠荷瘤骨肉瘤移植瘤后,能显著抑制肿瘤生长,瘤重的抑制率可达52%,免疫组化结果显示Ad-IL-24重组腺病毒能明显上调与细胞...     

Synthetic retinoid CD437 promotes rapid apoptosis in malignant human epidermal keratinocytes and G1 arrest in their normal counterparts

Four human cutaneous squamous cell carcinoma (SCC) cell lines and normal human epidermal keratinocyte (NHEK) cells from two donors were examined for sensitivity to the synthetic retinoid 6-[3-(1 -adamantyl)-4-hydroxyphenyl]-2-naph-thalene carboxylic acid (CD437) alone or in combination with other agents. CD437 promoted rapid (within 2 h) apoptosis in SCC cells and G1 arrest in NHEK cells. G1 arrest in NHEK cells was sustained for 48 h while apoptosis occurred in approximately 60% of SCC cell after 24 h. Apoptosis could not be inhibited by nuclear retinoic acid receptor antagonists or cycloheximide, indicating CD437 was functioning in a receptor-independent manner. All-trans retinoic acid not only failed to induce apoptosis in SCC cells even at 20-fold higher concentration relative to the effective concentration of CD437; it also decreased the efficacy of CD437. Because of its differential effects on normal versus malignant keratinocytes, CD437 may be useful for the prevention or treatment of cutaneous SCC.     

CD98 induces LFA-1-mediated cell adhesion in lymphoid cells via activation of Rap1

CD98 is a multifunctional heterodimeric membrane protein involved in the regulation of cell adhesion as well as amino acid transport. We show that CD98 cross-linking persistently activates Rap1 GTPase in a LFA-1-dependent manner and induces LFA-1/ICAM-1-mediated cell adhesion in lymphocytes. Specific phosphatidylinositol-3-kinase (PI3K) inhibitors suppressed both LFA-1 activation and Rap1GTP generation, and abrogation of Rap1GTP by retroviral over-expression of a specific Rap1 GTPase activating protein, SPA-1, totally inhibited the LFA-1/ICAM-1-mediated cell adhesion. These results suggest that CD98 cross-linking activates LFA-1 via the PI3K signaling pathway and induces accumulation of Rap1GTP in a LFA-1-dependent manner, which in turn mediates the cytoskeleton-dependent cell adhesion process.     

Identification and characterization of a cell cycle and apoptosis regulatory protein-1 as a novel mediator of apoptosis signaling by retinoid CD437

CD437, a novel retinoid, causes cell cycle arrest and apoptosis in a number of cancer cells including human breast carcinoma (HBC) by utilizing an undefined retinoic acid receptor/retinoid X receptor-independent mechanism. To delineate mediators of CD437 signaling, we utilized a random antisense-dependent functional knockout genetic approach. We identified a cDNA that encodes approximately 130-kDa HBC cell perinuclear protein (termed CARP-1). Treatments with CD437 or chemotherapeutic agent adriamycin, as well as serum deprivation of HBC cells, stimulate CARP-1 expression. Reduced levels of CARP-1 result in inhibition of apoptosis by CD437 or adriamycin, whereas increased expression of CARP-1 causes elevated levels of cyclin-dependent kinase inhibitor p21WAF1/CIP1 and apoptosis. CARP-1 interacts with 14-3-3 protein as well as causes reduced expression of cell cycle regulatory genes including c-Myc and cyclin B1. Loss of c-Myc sensitizes cells to apoptosis by CARP-1, whereas expression of c-Myc or 14-3-3 inhibits CARP-1-dependent apoptosis. Thus, apoptosis induction by CARP-1 involves sequestration of 14-3-3 and CARP-1-mediated altered expression of multiple cell cycle regulatory genes. Identification of CARP-1 as a key mediator of signaling by CD437 or adriamycin allows for delineation of pathways that, in turn, may prove beneficial for design and targeting of novel antitumor agents.