Hsp27 knockdown using nucleotide-based therapies inhibit tumor growth and enhance chemotherapy in human bladder cancer cells

Heat shock protein 27 (Hsp27) is a cytoprotective chaperone that is phosphoactivated during cell stress that prevents aggregation and/or regulate activity and degradation of certain client proteins. Recent evidence suggests that Hsp27 may be involved in tumor progression and the development of treatment resistance in various tumors, including bladder cancer. The purpose of this study was to examine, both in vitro and in vivo, the effects of overexpression of Hsp27 and, correspondingly, the down-regulation of Hsp27 using small interfering (si) RNA and OGX-427, a second-generation antisense oligonucleotide targeting Hsp27. Hsp27 overexpression increased UMUC-3 cell growth and resistance to paclitaxel. Both OGX-427 and Hsp27 siRNA decreased Hsp27 protein and mRNA levels by >90% in a dose- and sequence-specific manner in human bladder cancer UMUC-3 cells. OGX-427 or Hsp27 siRNA treatment induced apoptosis and enhanced sensitivity to paclitaxel in UMUC-3 cells. In vivo, OGX-427 significantly inhibited tumor growth in mice, enhanced sensitivity to paclitaxel, and induced significantly higher levels of apoptosis compared with xenografts treated with control oligonucleotides. Collectively, these findings suggest that Hsp27 knockdown with OGX-427 and combined therapy with paclitaxel could be a novel strategy to inhibit the progression of bladder cancer.     

B7-H3 silencing increases paclitaxel sensitivity by abrogating Jak2/Stat3 phosphorylation

In many types of cancer, the expression of the immunoregulatory protein B7-H3 has been associated with poor prognosis. Previously, we observed a link between B7-H3 and tumor cell migration and invasion, and in present study, we have investigated the role of B7-H3 in chemoresistance in breast cancer. We observed that silencing of B7-H3, via stable short hairpin RNA or transient short interfering RNA transfection, increased the sensitivity of multiple human breast cancer cell lines to paclitaxel as a result of enhanced drug-induced apoptosis. Overexpression of B7-H3 made the cancer cells more resistant to the drug. Next, we investigated the mechanisms behind B7-H3-mediated paclitaxel resistance and found that the level of Stat3 Tyr705 phosphorylation was decreased in B7-H3 knockdown cells along with the expression of its direct downstream targets Mcl-1 and survivin. The phosphorylation of Janus kinase 2 (Jak2), an upstream molecule of Stat3, was also significantly decreased. In contrast, reexpression of B7-H3 in B7-H3 knockdown and low B7-H3 expressing cells increased the phosphorylation of Jak2 and Stat3. In vivo animal experiments showed that B7-H3 knockdown tumors displayed a slower growth rate than the control xenografts. Importantly, paclitaxel treatment showed a strong antitumor activity in the mice with B7-H3 knockdown tumors, but only a marginal effect in the control group. Taken together, our data show that in breast cancer cells, B7-H3 induces paclitaxel resistance, at least partially by interfering with Jak2/Stat3 pathway. These results provide novel insight into the function of B7-H3 and encourage the design and testing of approaches targeting this protein and its partners.     

Targeting heterogeneous nuclear ribonucleoparticule A1 and A2 proteins by RNA interference promotes cell death in transformed but not in normal mouse cell lines

The heterogeneous nuclear ribonucleoparticule A1 and A2 proteins can bind to vertebrate single-stranded telomeric sequences. Moreover, changes in the levels of heterogeneous nuclear ribonucleoparticule A1 can influence telomere length in mouse and human cells. We have shown previously that the combined knockdown of A1 and A2 proteins in human transformed cells promotes apoptosis. In contrast, a similar reduction in A1 and A2 expression in normal mortal human cell lines does not induce cell death. Here, we show that a variety of mouse cell lines display a similar behavior on reduction of A1 and A2 protein levels using small interfering RNA. In addition, the expression of the mouse A1 cDNA protects human HeLa cells from apoptosis when human A1 and A2 proteins are targeted by RNA interference. Lastly, we show that knockdown of A1 and A2 expression also impairs the growth of a human transformed cell line that does not express telomerase. These results firmly establish A1 and A2 as proteins required for the viability of transformed murine and human cells, irrespective of the status of telomerase expression or the length of the double-stranded telomeric repeat.     

Synergistic suppression of prostatic cancer cells by coexpression of both murine double minute 2 small interfering RNA and wild-type p53 gene in vitro and in vivo

Our objective was to evaluate cell growth and death effects by inhibiting Murine Double Minute 2 (MDM2) expression in human prostate cancer cells overexpressing the wild-type (WT) p53 gene. Prostate PC-3 tumor cells were transfected with a plasmid containing either mdm2 small interfering (Si-mdm2) or the WT p53 gene (Pp53) alone, or both (Pmp53), using Lipofectamine in vitro and attenuated Salmonella enterica serovar Typhi vaccine strain Ty21a (Salmonella Typhi Ty21a) in vivo. Cell growth, apoptosis, and the expression of related genes and proteins were examined in vitro and in vivo by flow cytometry and Western blot assays. We demonstrated that human prostate tumors had increased expression of MDM2 and mutant p53 proteins. Transfection of the PC-3 cells with the Pmp53 plasmid in vitro offered significant inhibition of cell growth and an increase in apoptotic cell death compared with that of the Si-mdm2 or Pp53 group. These effects were associated with up-regulation of p21 and down-regulation of hypoxia-inducible factor 1α expression in Pmp53-transfected cells. To validate the in vitro findings, the nude mice implanted with PC-3 cells were treated with attenuated Salmonella Typhi Ty21a carrying the plasmids, which showed that the Pmp53 plasmid significantly inhibited the tumor growth rate in vivo compared with that of the Si-mdm2 or Pp53 plasmid alone. Tumor tissues from mice treated with the Pmp53 plasmid showed increased expression of p21 and decreased expression of hypoxia-inducible factor 1α proteins, with an increased apoptotic effect. These results suggest that knockdown of mdm2 expression by its specific small interfering RNA with overexpression of the WT p53 gene offers synergistic inhibition of prostate cancer cell growth in vitro and in vivo.     

Small interfering RNA-mediated knockdown of PRL phosphatases results in altered Akt phosphorylation and reduced clonogenicity of pancreatic cancer cells

The PRL phosphatases have been implicated in cancer cell growth and metastasis in a variety of tumor types. Using cDNA microarray, we previously identified and reported PRL-1 as being highly up-regulated in pancreatic cancer cell lines. In this study, we sought to further evaluate the expression of all three PRL phosphatases in pancreatic cancer cell lines and extend our findings to in situ analysis of primary pancreatic tumors taken directly from patients. Additionally, we determine if small interfering RNA-mediated knockdown of relevant PRLs confers antitumor effects in pancreatic cancer cells. Using oligonucleotide expression arrays, mRNA levels of PRL-1 and PRL-2 but not PRL-3 were identified as up-regulated in pancreatic cancer cell lines and tumor samples taken directly from patients compared with those of normal pancreas. Focusing on PRL-1 and PRL-2, high levels of both proteins were detected in a subset of pancreatic cancer cell lines and tumor samples using Western blotting and immunohistochemistry, respectively. Small interfering RNA-mediated knockdown of PRL-1 and PRL-2 in combination resulted in a moderate reduction of cellular growth and migration in MIA PaCa-2 and PANC-1 cells. More importantly, knockdown of both PRL-1 and PRL-2 significantly inhibited colony formation of these cells in soft agar as well as serum-induced Akt phosphorylation. These data support the hypothesis that PRL phosphatases regulate key pathways involved in tumorigenesis and metastasis and that knockdown of both PRL-1 and PRL-2 is required to disrupt PRL phosphatase function in pancreatic cancer cells.     

体外合成特异siRNA及其对Hela细胞端粒酶基因表达抑制的研究

目的 针对端粒酶蛋白催化亚单位(hTERT)基因不同片断合成2条siRNA,比较其对Hela细胞端粒酶基因表达干扰作用的强弱,从而选择出更好的干扰靶位点.方法 用T7RNA聚合酶在体外转录合成siRNA,将合成的2段siRNA转染Hela细胞,对其干扰作用进行分析、鉴定.结果 用2段合成的特异siRNA转染Hela细胞...     

人端粒酶反转录酶小发夹RNA质粒表达载体的构建及鉴定

背景:端粒酶反转录酶是端粒酶的活性亚基,已成为肿瘤研究的热点。RNA干扰技术作为一种基因沉默方法,具有高效、特异等优点,现已广泛应用于肿瘤、病毒等研究领域。目的:构建针对人端粒酶反转录酶的小发夹RNA质粒表达载体,并观察其对乳腺癌T47D细胞人端粒酶反转录酶基因的表达和端粒酶活性的影响。方法:以Genbank中人端粒酶反转录酶基因的mRNA序列为基础,设计人端粒酶反转录酶基因的小干扰RNA序列,将其连接到具有G418抗性的质粒pBAsi-hU6-Neo(BamHⅠ/HindⅢ)中,应用基因测序加以验证,扩增提取质粒,以脂质体转染表达小发夹RNA的质粒到乳腺癌T47D细胞,抗生素G418筛选出转染成功的各组细胞。结果与结论:实验所构建的人端粒酶反转录酶的小发夹RNA质粒表达载体,经测序验证无误。将pBAsi-hU6-Neo重组质粒转染入T47D细胞,经G418筛选获得了转染成功的细胞。经RT-PCR和Western blot检测,转染后的人端粒酶反转录酶基因在mRNA和蛋白水平的表达均明显降低(P<0.01),经TRAP-ELISA法检测实验组细胞端粒酶活性出现显著下降(P<0.01)。结果证实,实验成功构建人端粒酶反转录酶的小发夹RNA质粒表达载体,实验所设计的小干扰RNA能有效抑制肿瘤细胞人端粒酶反转录酶基因的表达,进而降低细胞的端粒酶活性。     

Treatment of bladder cancer cells in vitro and in vivo with 2-5A antisense telomerase RNA

Bladder cancer is the most common malignant tumor of the urinary tract. Novel treatment approaches are essential because of the failure of current treatment options to cure a high percentage of patients. Telomerase, a ribonucleoprotein, is detected in almost all bladder cancer, but not in normal bladder tissues. Therefore, telomerase is expected to be a very promising candidate for targeted therapy of bladder cancer. In this study, we synthesized a 19-mer antisense oligonucleotide against the RNA component of human telomerase (hTR) linked to a 2-5A molecule (2-5A-anti-hTR) and investigated its antitumor effect against bladder cancer cells. The 2-5A antisense strategy relies on the recruitment and activation of RNase L at the site of targeted RNA sequence. Here we demonstrate that treatment with 2-5A-anti-hTR reduced the viability of seven bladder cancer cell lines (UM-UC-2, UM-UC-3, UM-UC-6, UM-UC-9, UM-UC-14, RT4 and T24) expressing telomerase activity to 21-55% within 4 days. The cytotoxicity was mainly due to induction of caspase-dependent apoptosis. In contrast, normal fibroblast WI38 cells lacking telomerase activity were resistant to the treatment. Furthermore, treatment of subcutaneous UM-UC-2 tumors in nude mice with 2-5A-anti-hTR significantly suppressed the tumor growth through induction of apoptosis (P < 0.001). These findings may offer a strong support to the feasibility of the 2-5A-anti-hTR treatment for human bladder cancer.     

Dose-dependent modulatory effects of insulin on glucose-induced endothelial senescence in vitro and in vivo: a relationship between telomeres and nitric oxide

The elderly are prone to postprandial hyperglycemia that increases their cardiovascular risk. Although insulin therapy is necessary to treat diabetes, high plasma concentrations of insulin may cause the development of atherosclerosis and accelerate endothelial senescence. We assumed that high glucose causes stress-induced premature senescence and replicative senescence and examined the regulatory role of insulin in endothelial senescence and functions under different glucose conditions. Exposure of human endothelial cells to high glucose (22 mM) for 3 days increased senescence-associated-β-galactosidase activity, a senescence marker, and decreased telomerase activity, a replicative senescence marker. Physiological concentrations of insulin preserved telomere length and delayed endothelial senescence under high-glucose conditions. The effect of insulin under high-glucose conditions was associated with reduced reactive oxygen species and increased nitric oxide (NO). Small interfering RNA targeting endothelial NO synthase reduced the antisenescence effects of insulin. Physiological concentrations of insulin also reversed high glucose-induced increases in p53 and vascular cell adhesion molecule-1 and decreases in senescence marker protein-30. On the other hand, when insulin was given at any concentrations under normal glucose or at high concentrations under high glucose, its ability to promote cellular senescence was unrelated to endothelial NO. Finally, streptozotocin-induced diabetes showed more senescent cells in the aortic endothelium of aged rats compared with age-matched control and insulin-treated animals. Conclusively, the regulatory effects of insulin on endothelial senescence were modulated by the glucose environment. These data may help explain insulin's complicated roles in atherosclerosis in the elderly.     

The telomerase inhibitor PinX1 is a major haploinsufficient tumor suppressor essential for chromosome stability in mice

Telomerase is activated in most human cancers and is critical for cancer cell growth. However, little is known about the significance of telomerase activation in chromosome instability and cancer initiation. The gene encoding the potent endogenous telomerase inhibitor PinX1 (PIN2/TRF1-interacting, telomerase inhibitor 1) is located at human chromosome 8p23, a region frequently exhibiting heterozygosity in many common human cancers, but the function or functions of PinX1 in development and tumorigenesis are unknown. Here we have shown that PinX1 is a haploinsufficient tumor suppressor essential for chromosome stability in mice. We found that PinX1 expression was reduced in most human breast cancer tissues and cell lines. Furthermore, PinX1 heterozygosity and PinX1 knockdown in mouse embryonic fibroblasts activated telomerase and led to concomitant telomerase-dependent chromosomal instability. Moreover, while PinX1-null mice were embryonic lethal, most PinX1+/- mice spontaneously developed malignant tumors with evidence of chromosome instability. Notably, most PinX1 mutant tumors were carcinomas and shared tissues of origin with human cancer types linked to 8p23. PinX1 knockout also shifted the tumor spectrum of p53 mutant mice from lymphoma toward epithelial carcinomas. Thus, PinX1 is a major haploinsufficient tumor suppressor essential for maintaining telomerase activity and chromosome stability. These findings uncover what we believe to be a novel role for PinX1 and telomerase in chromosome instability and cancer initiation and suggest that telomerase inhibition may be potentially used to treat cancers that overexpress telomerase.     

Enhanced Therapeutic Efficacy by Simultaneously Targeting Two Genetic Defects in Tumors

Targeting tumor-specific gene abnormalities has become an attractive approach in developing therapeutics to treat cancer. Overexpression of Bcl2 and mutations of p53 represent two of the most common molecular defects in tumors. In the nucleus, p53 induces cell cycle arrest, while it interacts with Bcl2 outside of the nucleus to regulate signal pathways involved in apoptosis. To potentiate antitumor activity, we tested a “double target” approach to antitumor therapy by combining H101, a recombinant oncolytic adenovirus that targets the inactive p53 in tumors, with a small interfering RNA (siBCL2) that targets Bcl2. In cell culture, the combined treatment significantly enhanced apoptosis and cytotoxicity as compared with treatment with either H101 or siBCL2 alone. In animals carrying tumor xenographs, combined H101 and siBCL2 treatment significantly inhibited tumor growth and prolonged survival. At the end of the study, all animals in the combined therapy group survived and two of the five animals showed complete eradication of their tumors. Interestingly, siBCL2 treatment increased H101 viral replication in both treated cells and tumor tissues. Simultaneously targeting two tumor-specific gene abnormalities using an oncolytic adenovirus and siRNA potentiates total antitumor activity.     

Rac1基NRNAi慢病毒载体的构建与鉴定

背景：Rac1是通过信号转导来调节癌细胞的侵袭、增殖,它在各种肿瘤中都有表达。目的：构建RAC1基因RNA干扰慢病毒载体,并检测其干扰效率。方法：应用Western blot检测Rac1基因在舌癌细胞中的表达。针对筛选确定的Rac1基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经AgeI和EcoRI双酶切后的pMagic4.1载体连接产生短发卡RNA慢病毒载体,PCR筛选阳性克隆,测序鉴定后将质粒分别和包装质粒混合物共转染293T细胞,采用Real-Time PCR筛靶,将筛靶成功的质粒进行慢病毒包装,再感染Tca8113细胞。荧光定量PCR和Western blot检测鉴定Rac1基因表达的干扰效果以确定其生物活性。结果与结论：成功构建了RAC1干扰载体,Western blot检测显著抑制Rac1蛋白表达,经Real-time PCR检测pLVT447的抑制率达70%。成功的构建了Rac1基因RNAi慢病毒载体,为RNAi用于靶向Rac1的基因治疗舌癌（Tca8113）提供了有效的siRNA靶序列。     

Small interfering RNA targeting of S phase kinase-interacting protein 2 inhibits cell growth of oral cancer cells by inhibiting p27 degradation

S phase kinase-interacting protein 2 (Skp2), an F box protein, is required for the ubiquitination and consequent degradation of p27. It is well known that reduced expression of p27 is frequently observed in various cancers including oral squamous cell carcinoma and is due to an enhancement of its protein degradation. Our previous study showed that overexpression of Skp2 was frequently found in oral squamous cell carcinoma and inversely correlated with p27 expression. Recently, a technique known as RNA interference has been successfully adapted to mammalian cells. In the present study, we investigated if small interfering RNA (siRNA)-mediated gene silencing of Skp2 can be employed in order to inhibit p27 down-regulation in oral squamous cell carcinoma. We used a siRNA plasmid vector, which has an advantage over synthetic siRNAs in determining the effects of decreasing the high constitutive levels of Skp2 protein in oral squamous cell carcinoma. We showed that Skp2 siRNA transfection decreased Skp2 protein and induced the accumulation of p27 protein in oral squamous cell carcinoma cells. Moreover, p27 protein in Skp2 siRNA-transfected cells is more stabilized than that in control siRNA-transfected cells. Interestingly, Skp2 siRNA inhibited the cell proliferation of oral squamous cell carcinoma cells both in vitro and in vivo. Our findings suggest that siRNA-mediated gene silencing of Skp2 can be a novel modality of cancer gene therapy for suppression of p27 down-regulation.     

eIF4E knockdown decreases breast cancer cell growth without activating Akt signaling

Activation of translation initiation is essential for the malignant phenotype and is emerging as a potential therapeutic target. Translation is regulated by the expression of translation initiation factor 4E (eIF4E) as well as the interaction of eIF4E with eIF4E-binding proteins (e.g., 4E-BP1). Rapamycin inhibits translation initiation by decreasing the phosphorylation of 4E-BP1, increasing eIF4E/4E-BP1 interaction. However, rapamycin also inhibits S6K phosphorylation, leading to feedback loop activation of Akt. We hypothesized that targeting eIF4E directly would inhibit breast cancer cell growth without activating Akt. We showed that eIF4E is ubiquitously expressed in breast cancer cell lines. eIF4E knockdown by small interfering RNA inhibited growth in different breast cancer cell subtypes including triple-negative (estrogen receptor/progesterone receptor/HER-2-negative) cancer cells. eIF4E knockdown inhibited the growth of cells with varying total and phosphorylated 4E-BP1 levels and inhibited rapamycin-insensitive as well as rapamycin-sensitive cell lines. eIF4E knockdown led to a decrease in expression of cyclin D1, Bcl-2, and Bcl-xL. eIF4E knockdown did not lead to Akt phosphorylation but did decrease 4E-BP1 expression. We conclude that eIF4E is a promising target for breast cancer therapy. eIF4E-targeted therapy may be efficacious in a variety of breast cancer subtypes including triple-negative tumors for which currently there are no targeted therapies. Unlike rapamycin and its analogues, eIF4E knockdown is not associated with Akt activation.     

Docetaxel-induced apoptosis in melanoma cells is dependent on activation of caspase-2

Taxanes have a broad spectrum of activity against various human cancers, including melanoma. In this study, we have examined the molecular mechanism of docetaxel-induced apoptosis of human melanoma. We report that docetaxel induced varying degrees of apoptosis in a panel of melanoma cell lines but not in normal fibroblasts. Induction of apoptosis was caspase dependent and associated with changes in mitochondrial membrane potential that could be inhibited by overexpression of Bcl-2. Docetaxel induced changes in Bax that correlated with sensitivity to docetaxel-induced apoptosis. These changes in Bax were not inhibited by overexpression of Bcl-2. Kinetic studies of caspase-2 activation by Western blotting and fluorogenic assays revealed that activation of caspase-2 seemed to be the initiating event. Inhibition of caspase-2 with z-VDVAD-fmk or by small interfering RNA knockdown inhibited changes in Bax and mitochondrial membrane potential and events downstream of mitochondria. Activation of caspase-8 and Bid seemed to be a late event, and docetaxel was able to induce apoptosis in cells deficient in caspase-8 and Bid. p53 did not seem to be involved as a p53 null cell line was sensitive to docetaxel and an inhibitor of p53 did not inhibit apoptosis. Small interfering RNA knockdown of PUMA and Noxa also did not inhibit apoptosis. These results suggest that docetaxel induces apoptosis in melanoma cells by pathways that are dependent on activation of caspase-2, which initiates mitochondrial dependent apoptosis by direct or indirect activation of Bax.