The novel histone deacetylase inhibitor,AR‐42, inhibits gp130/Stat3 pathway and induces apoptosis and cell cycle arrest in multiple myeloma cells

Multiple myeloma (MM) remains incurable with current therapy, indicating the need for continued development of novel therapeutic agents. We evaluated the activity of a novel phenylbutyrate‐derived histone deacetylase inhibitor, AR‐42, in primary human myeloma cells and cell lines. AR‐42 was cytotoxic to MM cells at a mean LC₅₀ of 0.18 ± 0.06 μmol/l at 48 hr and induced apoptosis with cleavage of caspases 8, 9 and 3, with cell death largely prevented by caspase inhibition. AR‐42 downregulated the expression of gp130 and inhibited activation of STAT3, with minimal effects on the PI3K/Akt and MAPK pathways, indicating a predominant effect on the gp130/STAT‐3 pathway. AR‐42 also inhibited interleukin (IL)‐6‐induced STAT3 activation, which could not be overcome by exogenous IL‐6. AR‐42 also downregulated the expression of STAT3‐regulated targets, including Bcl‐xL and cyclin D1. Overexpression of Bcl‐xL by a lentivirus construct partly protected against cell death induced by AR‐42. The cyclin dependent kinase inhibitors, p16 and p21, were also significantly induced by AR‐42, which together with a decrease in cyclin D1, resulted in G₁ and G₂ cell cycle arrest. In conclusion, AR‐42 has potent cytotoxicity against MM cells mainly through gp130/STAT‐3 pathway. The results provide rationale for clinical investigation of AR‐42 in MM.     

Role of MIF/CXCL8/CXCR2 signaling in the growth of nasopharyngeal carcinoma tumor spheres

Macrophage migration inhibitory factor (MIF) and CXCL8 (also named IL-8) are strongly expressed in the tissues of nasopharyngeal carcinoma (NPC). However, their role in the growth of NPC has not been fully examined. This study aims to evaluate the functions of MIF and CXCL8 on the growth of NPC tumor spheres. The elevated expression of CXCL8 in tumor over normal tissues was confirmed in 37 pairs of biopsies from NPC patients. In the in vitro study, all the poorly differentiated NPC cell lines, including the EBV-positive C666-1, and the EBV-negative CNE-1, CNE-2, SUNE-1, HNE-1 and HONE-1 cells, were found to express CXCL8 and MIF. Therefore, the EBV-positive C666-1 cell was selected to examine for the role of MIF and CXCL8 in the growth of the NPC tumor spheres. Functional study showed that the growth of C666-1 tumor spheres, under the nutrient poor or growth factor supplemented culture conditions, could be inhibited by the CXCL8 specific peptide inhibitor. The growth of the tumor spheres could also be reduced by the CXCR2 specific inhibitor SB225002 or the PI3K/AKT inhibitor LY294002, indicating that the endogenously produced CXCL8 plays an autocrine role in the growth of the tumor spheres. Further mechanistic studies revealed that the gene expression of CXCL8 could be reduced by the MIF specific small interfering RNA (siRNA) or NF-κB inhibitor parthenolide, and the growth of tumor spheres was also reduced after MIF siRNA transfection. Taken together, the present study highlights the role of MIF/CXCL8/CXCR2 axis in the growth of NPC tumor spheres. Chemotherapeutic interference of this signaling pathway may help to control the growth of the NPC tumor.     

Silencing of hypoxia-inducible tumor suppressor lysyl oxidase gene by promoter methylation activates carbonic anhydrase IX in nasopharyngeal carcinoma

Lysyl oxidase (LOX) is an oxidative enzyme known to initiate the cross-linking of collagens and elastin, and suggested recently as a tumor suppressor for several tumor types including lung, pancreatic and gastric cancers. Previously we showed that LOX is strongly induced upon hypoxia in nasopharyngeal carcinoma (NPC) cell lines CNE2 and HONE1 but only slightly in HK1 and not in C666-1. Here, we further studied the regulatory mechanism and functions of LOX in NPC. LOX is widely expressed in human normal tissues with variations in expression levels. LOX was expressed in most NPC cell lines except for C666-1, while HK1 and FaDu (laryngeal cancer) only expressed low level of LOX. Methylation analysis showed that the LOX promoter was methylated in C666-1 and partially methylated in HK1. After demethylation with 5-aza-2’-deoxycytidine, LOX expression was reactivated along with increased unmethylated alleles. LOX promoter methylation was detected in 42/49 (85.7%) of NPC primary tumors but only 3/16 (18.75%) of nose swab samples from NPC patients. LOX overexpression reduced the clonogenicity and cell growth of NPC cells, and also inhibited the migration and invasion of the NPC cells. Carbonic anhydrase IX (CA9) mRNA level was obviously decreased in HK1 cells after transfection with LOX. The elevation of CA9 protein upon hypoxia was inhibited in LOX-transfected HK1 cells. The protein levels of an apoptosis marker cPARP were increased in LOX-transfected HK1 cells upon hypoxia treatment. Our data showed that silencing or down-regulation of LOX in NPC was due to its promoter methylation and LOX acts as a tumor suppressor in NPC. LOX silencing would facilitate NPC cells to escape from hypoxia-induced apoptosis and maintains a malignant and metastatic phenotype.     

The Bcl‐xL inhibitor of apoptosis is preferentially expressed in cutaneous squamous cell carcinoma compared with that in keratoacanthoma

Keratoacanthoma (KA) is difficult to histologically distinguish from squamous cell carcinoma (SCC). Therefore, although KA is a benign self‐resolving skin lesion, KA is commonly treated as SCC. Biomarkers to distinguish KA and SCC would thus be desirable. In search for specific markers, paraffin‐embedded tissue samples from 25 SCC and 64 KA were arranged in a tissue microarray (TMA) and stained for immunologic cell‐markers CD3, CD20 and CD68 as well as for proteins considered of relevance in tumorgenesis, namely NFκB/p65, IκB‐α, STAT3, p53, TRAP‐1, pRB, phosphorylated pRb, Cyld, p21, p16^INK4, Survivin, Bcl‐xL, Caspase 3, Bak, FLK‐1/VEGF‐r2 and Ki‐67. In addition, the tumors were tested for presence of human papillomavirus by PCR. We detected that the two lesions differed significantly in expression of Bcl‐xL which was present in 84% of the SCC compared with only 15% in the KA (p < 0.001). The lower expression of the antiapoptotic protein Bcl‐xL in KA is consistent with a possible role of apoptosis in the regression of KA. © 2008 Wiley‐Liss, Inc.     

miR‐185‐3p regulates nasopharyngeal carcinoma radioresistance by targeting WNT2B in vitro

Aberrant microRNA (miRNA) expression contributes to a series of malignant cancer behaviors, including radioresistance. Our previous study showed differential expression of miR‐185‐3p in post‐radiation nasopharyngeal carcinoma (NPC) cells. To investigate the role of miR‐185‐3p in NPC radioresistance, CNE‐2 and 5‐8F cells were transfected with miR‐185‐3p mimic and miR‐185‐3p inhibitor, respectively. CCK‐8 assay and colony formation experiment confirmed that the expression of miR‐185‐3p affected the radioresistance of NPC cells. A negative correlation between miR‐185‐3p and WNT2B expression was observed in NPC cells and tissues. Luciferase reporter assays confirmed that miR‐185‐3p directly targeted the coding region of WNT2B. Furthermore, we found radioresistance decreased in WNT2B‐silenced NPC cells. Activation of the WNT2B/β‐catenin pathway was accompanied by epithelial–mesenchymal transition biomarker changes in NPC. We concluded that miR‐185‐3p contributed to the radioresistance of NPC via modulation of WNT2B expression in vitro.     

ApoG2, a novel inhibitor of antiapoptotic Bcl-2 family proteins, induces apoptosis and suppresses tumor growth in nasopharyngeal carcinoma xenografts

Nasopharyngeal carcinoma (NPC) is a common malignant tumor in South China. It has been reported that overexpression of antiapoptotic Bcl-2 family proteins in NPC has caused the lack of long-term efficacy of conventional therapies. Apogossypolone (ApoG2), a novel small-molecule inhibitor of antiapoptotic Bcl-2 family proteins, has been discovered as the optimized derivative of gossypol. In this study, we found that in NPC cells, ApoG2 totally blocked the antiapoptotic function of Bcl-2 family proteins without affecting the expression levels of these proteins. ApoG2 selectively inhibited proliferation of 3 NPC cell lines (C666-1, CNE-1 and CNE-2) that highly expressed the antiapoptotic Bcl-2 proteins. This inhibitory activity was associated with release of cytochrome c, activation of caspase-9 and caspase-3 and apoptosis of sensitive NPC cells. However, ApoG2 had no obvious inhibitory effect on NPC cell line HONE-1, which expressed antiapoptotic Bcl-2 and Bcl-xL at a low level. We further found that ApoG2 effectively suppressed tumor growth of NPC xenografts in nude mice and enhanced the antitumor effect of CDDP (cisplatin) on NPC cells in vitro and in vivo. Immunohistochemical results showed that the expression of CD31 decreased after ApoG2 treatment, which suggested inhibition of angiogenesis in NPC xenografts. Our findings strongly suggest that ApoG2 may serve as a novel inhibitor of Bcl-2 family proteins and, by targeting these proteins, may become a promising drug for the treatment of NPC.     

TM‐233, a novel analog of 1′‐acetoxychavicol acetate,induces cell death in myeloma cells by inhibiting both JAK/STAT and proteasome activities

Although the introduction of bortezomib and immunomodulatory drugs has led to improved outcomes in patients with multiple myeloma, the disease remains incurable. In an effort to identify more potent and well‐tolerated agents for myeloma, we have previously reported that 1′‐acetoxychavicol acetate (ACA), a natural condiment from South‐East Asia, induces apoptotic cell death of myeloma cells in vitro and in vivo through inhibition of NF‐κB‐related functions. Searching for more potent NF‐κB inhibitors, we developed several ACA analogs based on quantitative structure–activity relationship analysis. TM‐233, one of these ACA analogs, inhibited cellular proliferation and induced cell death in various myeloma cell lines with a lower IC₅₀ than ACA. Treatment with TM‐233 inhibited constitutive activation of JAK2 and STAT3, and then downregulated the expression of anti‐apoptotic Mcl‐1 protein, but not Bcl‐2 and Bcl‐xL proteins. In addition, TM‐233 rapidly decreased the nuclear expression of NF‐κB and also decreased the accumulation of cytosolic NF‐κB. We also examined the effects of TM‐233 on bortezomib‐resistant myeloma cells that we recently established, KMS‐11/BTZ and OPM‐2/BTZ. TM‐233, but not bortezomib, inhibited cellular proliferation and induced cell death in KMS‐11/BTZ and OPM‐2/BTZ cells. Interestingly, the combination of TM‐233 and bortezomib significantly induced cell death in these bortezomib‐resistant myeloma cells through inhibition of NF‐κB activity. These results indicate that TM‐233 could overcome bortezomib resistance in myeloma cells mediated through different mechanisms, possibly inhibiting the JAK/STAT pathway. In conclusion, TM‐233 might be a more potent NF‐κB inhibitor than ACA, and could overcome bortezomib resistance in myeloma cells.     

PPARb/d Agonist GW501516 Inhibits Tumorigenesis and Promotes Apoptosis of the Undifferentiated Nasopharyngeal Carcinoma C666-1 Cells by Regulating miR-206

In previous investigations, we reported that peroxisome proliferator-activated receptor / (PPAR / ) activation by GW501516 inhibits proliferation and promotes apoptosis in the undifferentiated C666-1 nasopharyngeal carcinoma (NPC) cells by modulating caspase-dependent apoptotic pathway. In the present study, the mechanism by which GW501516 induces apoptosis was explored from the perspective of microRNA (miRNA) expression. Among the assayed miRNAs that were involved in regulating the expression of antiapoptotic protein Bcl-2, miR-206 was increased significantly and specifically by GW501516 in C666-1 cells at both the in vitro level and at the in vivo xenograft samples. The induction on miR-206 expression caused by GW501516 was capable of being antagonized by the PPAR / antagonist GSK3787 and AMPK antagonist dorsomorphin in C666-1 cells. GW501516’s suppression on the growth and apoptosis of C666-1 cells was found to be dependent on the presence of miR-206. miR-206 overexpression resulted in suppressed proliferation and colony formation ability, and further triggered increased apoptosis in C666-1 cells in a caspase-dependent manner. The expression of cleaved caspase 3 and caspase 9, and the ratio of Bax to Bcl-2 were elevated remarkably by miR-206. Consistent with the in vitro result, miR-206 was corroborated to suppress the ectopic NPC xenograft tumorigenesis that derived from the C666-1 cells in BALB/c nu/nu mice. Taken together, the current data demonstrated that miR-206 plays a critical role in the direct apoptosis-promoting effect induced by GW501516 in C666-1 cells. Furthermore, the emphasized tumor-suppressive role of miR-206 in the C666-1 cells indicates that it has the potential to provide a new therapeutic approach for the undifferentiated NPC.     

Silencing of the retinoid response gene TIG1 by promoter hypermethylation in nasopharyngeal carcinoma

Tazarotene-induced gene 1 (TIG1) and Tazarotene-induced gene 3 (TIG3) are retinoid acid (RA) target genes as well as candidate tumor suppressor genes in human cancers. In our study, we have investigated the expression of TIG1 and TIG3 in nasopharyngeal carcinoma (NPC). Loss of TIG1 expression was found in 80% of NPC cell lines and 33% of xenografts, whereas TIG3 was expressed in all NPC samples and immortalized nasopharyngeal epithelial cells. In order to elucidate the epigenetic silencing of TIG1 in NPC, the methylation status of TIG1 promoter was examined by genomic bisulfite sequencing and methylation-specific PCR (MSP). We have detected dense methylation of TIG1 5'CpG island in the 5 TIG1-negative NPC cell lines and xenograft (C666-1, CNE1, CNE2, HONE1 and X666). Partial methylation was observed in 1 NPC cell line HK1 showing dramatic decreased in TIG1 expression. Promoter methylation was absent in 2 TIG1-expressed NPC xenografts and the normal epithelial cells. Restoration of TIG1 expression and unmethylated alleles were observed in NPC cell lines after 5-aza-2'-deoxycytidine treatment. Moreover, the methylated TIG1 sequence was detected in 39 of 43 (90.7%) primary NPC tumors by MSP. In conclusion, our results showed that TIG1 expression is lost in the majority of NPC cell lines and xenografts, while promoter hypermethylation is the major mechanism for TIG1 silencing. Furthermore, the frequent epigenetic inactivation of TIG1 in primary NPC tumors implied that it may play an important role in NPC tumorigenesis.     

C2orf40蛋白在鼻咽癌中的表达及意义

目的 探讨C2orf40蛋白在鼻咽癌组织和细胞中的表达及其与细胞分化、临床病理特征的关系.方法 收集2001年1月至2003年12月在汕头大学医学院附属肿瘤医院诊断为鼻咽癌的122例病理活检组织石蜡标本及其病历资料,另外收集25例慢性鼻咽炎的石蜡标本作为对照.采用免疫组织化学法检测C2orf40蛋白表达情况,同时分析C2orf40表达水平与临床病理特征的关系.体外实验应用Western blotting对鼻咽癌细胞株CNE1、CNE2和C666-1进行C2orf40蛋白表达的测定,分析C2orf40表达与细胞分化程度的关系.结果 C2or40在对照组的阳性率为96.0％(24/25),且88.0％为高表达,病例组阳性率为58.2％ (71/122),但82.0％为低表达,二者差异有统计学意义(U=255.500,P＜0.001).C2orf40表达与淋巴结转移分期(N分期)和临床分期呈负相关(r=-0.058,P＜0.001;r=-0.202,P=0.026).C2orf40在高分化鼻咽癌细胞株CNE1中呈高表达,在低分化鼻咽癌细胞株CNE2和C666-1中呈低表达.结论 C2orf40蛋白表达下调与肿瘤细胞分化、淋巴结转移和临床分期相关,是鼻咽癌发生、发展的分子事件之一,可能成为抗肿瘤治疗的潜在靶点.     

beta-Sitosterol induces G2/M arrest, endoreduplication, and apoptosis through the Bcl-2 and PI3K/Akt signaling pathways

β-Sitosterol (SITO) is a potentially valuable candidate for cancer chemotherapy, however the cellular and molecular mechanisms responsible for its anti-cancer activity are unknown. Therefore, we attempted to elucidate the mechanisms responsible for SITO-induced anti-proliferation in human leukemia cells. Treatment with SITO increased caspase-3 activation and DNA fragmentation in U937 and HL60 cells. This effect was associated with significant G₂/M arrest and endoreduplication. We also demonstrated that SITO treatment significantly increases levels of polymeric -tubulin and promoted microtubule polymerization. We next elucidated that ectopic expression of Bcl-2 accelerates endoreduplication in U937 cells. Furthermore, the specific Bcl-2 inhibitor, HA14-1, prevented endoreduplication through G₂ phase arrest. Interestingly, SITO treatment did not significantly promote endoreduplication or decrease cell viability in Bcl-2 null K562 cells. SITO treatment also induced a gradual increase of phosphatidyl-inositol 3-kinase (PI3K) and Akt phosphorylation. Treatment with the selective PI3K/Akt inhibitor LY29004 completely blocked endoreduplication and apoptosis in the presence of SITO. In addition, treatment with SITO-induced phosphorylation of extracellular signal-regulated protein kinase (ERK), however significance of ERK activation in the execution of apoptosis and endoreduplication is unknown. These results suggest that SITO induces endoreduplication by promoting spindle microtubule dynamics through the Bcl-2 and PI3K/Akt signaling pathways.     

Mechanisms of 2-methoxyestradiol-induced apoptosis and G2/M cell-cycle arrest of nasopharyngeal carcinoma cells

2-Methoxyestradiol (2ME2) is an endogenous metabolite of 17beta-estradiol (E(2)). This study aims to examine the anti-tumour activities of 2ME2 on the poorly differentiated HONE-1 NPC cell line. At the concentration of 1muM, 2ME2 was found to induce a short-term reversible G2/M cell-cycle arrest. Further 10-fold increase to 10muM, 2ME2 induced both irreversible G2/M phase cell-cycle arrest and apoptosis. Induction of apoptosis and G2/M cell-cycle arrest was due to oxidative stress as both apoptosis and the proportion of cells arresting at G2/M phase could be reduced by the superoxide dismutase (SOD) mimetic, TEMPO. Induction of apoptosis was accompanied with proteolytic cleavage of caspase-9 and -3, but not caspase-8. Kinetics studies revealed that 2ME2 induced a time-dependent inhibition of extracellular signal-regulated protein kinase (ERK) and an activation of c-jun N-terminal kinases (JNKs). The chemical inhibitor of JNKs, SP600125, was found to reduce 2ME2-induced apoptosis of the HONE-1 cells. Confocal microscopy revealed that the induction of G2/M cell-cycle arrest was associated with the presence of immunoreactivity of p-cdc2 (Tyr15) in the nucleus. The G2/M cell-cycle arrest is also correlated with an increased level of inactive p-cdc25C (Ser216) in 2ME2-treated HONE-1 cells. Results from this study indicate that production of superoxide anions might be involved in 2ME2-induced apoptosis and G2/M cell-cycle arrest of the HONE-1 cells.     

Betulinic acid suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase SHP‐1 in human multiple myeloma cells

STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulate the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid‐induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP‐1 and silencing of the SHP‐1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescued betulinic acid‐induced cell death. Betulinic acid also downregulated the expression of STAT3‐regulated gene products such as bcl‐xL, bcl‐2, cyclin D1 and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub‐G1 cell population and an increase in caspase‐3‐induced PARP cleavage. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the betulinic acid‐induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10 to 55%) and bortezomib (from 5 to 70%) in MM cells. Overall, our results suggest that betulinic acid downregulates STAT3 activation through upregulation of SHP‐1, and this may have potential in sensitization of STAT3 overexpressing tumors to chemotherapeutic agents.     

2‐Methoxyestradiol induced bax phosphorylation and apoptosis in human retinoblastoma cells via p38 MAPK activation

Retinoblastoma (Rb) is a common childhood intraocular cancer that affects approximately 300 children each year in the United States alone. 2‐Methoxyestradiol (2ME), an endogenous metabolite of 17‐β‐estradiol that dose not bind to nuclear estrogen receptor, exhibits potent apoptotic activity against rapidly growing tumor cells. Here, we report that 2ME induction of apoptosis was demonstrated by early fragmented DNA after 48 h of incubation with 10 µM 2ME in Rb cell lines. Subsequently, a decrease of proliferation was observed in a time‐ and dose‐dependent manner. Further analysis of the mechanism indicates that p38 kinase plays a critical role in 2ME‐induced apoptosis in Y79 cells, even though ERK was also activated by 2ME under the same conditions. Activation of p38 kinase also mediates 2ME induced Bax phosphorylated at Thr¹⁶⁷ after a 6 h treatment of 2ME, which in turn prevents formation of the Bcl‐2‐Bax heterodimer. Both p38 specific inhibitor, SB 203580, or p38 knockdown by specific siRNA, blocked 2ME induction of Bax phosphorylation. Furthermore, only transiently transfected mutant BaxT167A, but not Bax S163A, inhibited 2ME‐induced apoptosis. In summary, our data suggest that 2ME induces apoptosis in human Rb cells by causing phosphorylation of p38 Mitogen‐activated protein kinase (MAPK), which appears to be correlated with phosphorlation of Bax. This understanding of 2ME's ability may help develop it as a promising therapeutic candidate by inducing apoptosis in a Rb. © 2011 Wiley Periodicals, Inc.