[Show abstract][Hide abstract] ABSTRACT: Despite of tremendous research efforts to profile prostate cancer, the genetic alterations and biological processes that correlate with disease progression remain partially elusive. In this study we show that the STAT3 small molecule inhibitor Stattic caused S-phase accumulation at low-dose levels and led to massive apoptosis at a relatively high-dose level in prostate cancer cells. STAT3 knockdown led to the disruption of the microvascular niche which tumor-initiating cells (TICs) and non-tumor initiating cells (non-TICs)depend on. Primary human prostate cancer cells and prostate cancer cell line contained high aldehyde dehydrogenase activity (ALDHhigh) subpopulations with stem cell-like characteristics, which expressed higher levels of the active phosphorylated form of STAT3 (pSTAT3) than that of non-ALDHhigh subpopulations. Stattic could singnificantly decreas the population of ALDHhigh prostate cancer cells even at low-dose levels. IL-6 can convert non-ALDHhigh cells to ALDHhigh cells in prostate cancer cell line as well as from cells derived from human prostate tumors, the conversion mediated by IL-6 was abrogated in the presence of STAT3 inhibitor or upon STAT3 knockdown. STAT3 knockdown significantly impaired the ability of prostate cancer cells to initiate development of prostate adenocarcinoma. Moreover, blockade of STAT3 signaling was significantly effective in eradicating the tumor-initiating and bulk tumor cancer cell populations in both prostate cancer cell-line xenograft model and patient-derived tumor xenograft (PDTX) models. This data suggests that targeting both tumor initiating and differentiated cell populations by STAT3 inhibition is predicted to have greater efficacy for prostate cancer treatment.
[Show abstract][Hide abstract] ABSTRACT: Glucose-regulated protein 78 (GRP78), the most abundant and well-characterized glucose-regulated protein, is a major stress-inducible chaperone localized to the endoplasmic reticulum (ER). The purpose of the present study was to investigate the mechanisms of GRP78 involved in the senescence sensitivity of ovarian cancer cells to cisplatin. In the present study, we found that the chemotherapy-sensitive ovarian tumor sections showed strong staining for heterochromatin protein 1-γ (HP1-γ), but weak staining for GRP78. Cisplatin-sensitive A2780 cells with low expression of GRP78 tended to undergo senescence easily when compared with the cisplatin-resistant C13K cells following a dose-gradient cisplatin exposure. Forced overexpression of GRP78 protected the cisplatin-sensitive A2780 cells from cisplatin-induced senescence through P53 and CDC2. Knockdown of GRP78 rescued the senescence sensitivity of cisplatin-resistant C13K cells to cisplatin through P21 and CDC2. Twisting of Ca2+ release from ER stores by GRP78 was established to be associated with the sensitivity of cisplatin-induced senescence in ovarian cancer cell lines. In conclusion, GRP78 may have anti-senescence effects on ovarian cancer cells involving multiple mechanisms. Intervention against GRP78 may reduce cisplatin resistance in ovarian cancer.
[Show abstract][Hide abstract] ABSTRACT: Adenovirus 5 vectors, known respectively as, the first generation, second generation and oncolytic adenovirus, have been studied extensively in preclinical and clinical trials. However, hitherto few systemic evaluations of the efficacy and toxicity of these adenoviral vectors that have reflected the vertical history of adenovirus based cancer gene therapy strategies have been undertaken. This study has chosen Adv-TK, the well-established adjuvant treatment in cancer, and compared its efficacy and safety with those of the two newly synthesized oncolytic adenovirus vectors encoding the HSV-TK gene, namely M7 and M8. The results obtained showed that systemic administration of 1×108 pfu M7 had an anti-tumour efficacy similar to that of 3×1010 pfu Adv-TK whilst M8 performed even better. Furthermore, compared to Adv-TK, M7 and M8 reduced the incidence of metastases and substantially prolonged the survival time of the mice xenografted with human orthotopic gastric carcinomas with disseminated metastasis. Even more exciting, however, were the similar toxic and immune safety results obtained from the administration of high doses of M7 or M8 in comparison with Adv-TK in immunocompetent and permissive syrian hamster. The data here exhibit a comprehensive display of the efficacy and safety of the three mutants and provide evidence for the future preclinical use of the M7 and M8 viruses.
PLoS ONE 04/2014; 9(4):e94050. DOI:10.1371/journal.pone.0094050 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Accelerated senescence (ACS) leading to proliferative arrest is a physiological mechanism of the DNA damage response that occurs during tumor therapy. Our experiment was designed to detect unknown genes that may play important roles in cisplatin-induced senescence and to illustrate the related senescence mechanism. Using 2-dimension electrophoresis (2-DE), we identified 5 protein spots with different expression levels in the normal and senescent NG108-15 cells. According to MALDI-TOF MS analysis, the 5 proteins were determined to be peptidylprolyl isomerase A (PPIA), peroxiredoxin 1 (PRX1), glutathione S-transferase mu 1 (GSTM1), vimentin (VIM) and glucose-regulated protein 78 (GRP78). Then, we investigated how cisplatin-induced senescence was mediated by GRP78 in the NG108-15 cells. Knockdown of GRP78 significantly increased P53 expression in NG108-15 cells. Additionally, 2-deoxy-D-glucose (2DG)-induced GRP78 overexpression protected the NG108-15 cells from cisplatin-induced senescence, which was accompanied by the obvious suppression of P53 and p-CDC2 expression. Inhibition of Ca2+ release from endoplasmic reticulum (ER) stores was also found to be associated with the anti-senescence effect of 2DG-induced GRP78 overexpression. In conclusion, we found 5 proteins that were differentially expressed in normal NG108-15 cells and senescent NG108-15 cells. GRP78 plays an important role in cisplatin-induced senescence in NG108-15 cells, mainly through its regulation of P53 expression and ER calcium efflux.
PLoS ONE 03/2014; 9(3):e90114. DOI:10.1371/journal.pone.0090114 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of the present study was to investigate the role of Stat3 in cisplatin resistant ovarian cancer. It was first demonstrated that higher activated Stat3 was detected in cisplatin-resistant ovarian cancer cell lines. To provide evidence that supported the hypothesis that phosphorylated-Stat3 expression may promote cisplatin resistance, ectopic Stat3 was expressed by IL-6 stimulation that partially abrogates Stat3, as opposed to the knock-down of Stat3 by specific siRNA that restores cisplatin sensitivity against ovarian cancer cells. This hypothesis was further confirmed by clinical tumor specimens of ovarian cancer obtained from patients with cisplatin-resistance. Based on these premises, Stattic, an effective small molecular inhibitor of Stat3, was used to inhibit Stat3 activation. The data presented here show that Stattic restored the sensitivity to cisplatin in chemoresistant ovarian cancer by significant reductions in the expression of the anti-apoptosis protein Bcl-2, Bcl-xl, Survivin protein and phosphorylated-Akt levels. Consistent with these observations, this experiment demonstrated the first evidence of Stattic circumvented cisplatin resistance of orthotopic xenograft ovarian cancer in vivo. Altogether, these findings emphasize the importance of Stat3 in cisplatin resistance in ovarian cancer and provide a further impetus to clinically evaluate biological modifiers that may circumvent cisplatin resistance in patients with chemoresistant ovarian cancer.
Cancer letters 08/2013; 341(2). DOI:10.1016/j.canlet.2013.08.022 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Several lines of evidence support an important role for Snail, a transcriptional factor, in breast cancer. Overexpression of Snail has been associated with breast cancer metastasis, although the specific role of Snail in the process remains unclear. To address this issue, the expression levels of Snail, RhoA and fibronectin, as well as MMP-2, were reduced in the breast tumor cell lines MDA-MB-231 and MDA-MB-435S, and their biological responses were studied in vitro and in vivo. For the first time, it was observed that downregulated Snail expression is correlated with a significant inhibition of the expression and activity of RhoA GTPase, as well as MMP-2. The present data provide evidence that Snail promotes tumor cell motility and angiogenesis which is mainly mediated through the regulation of RhoA activity. In conclusion, the present findings demonstrate a key regulatory role for Snail in breast tumor growth and progression.
[Show abstract][Hide abstract] ABSTRACT: Ovarian cancer is the leading cause of gynecologic cancer deaths among women. Although platinum-based chemotherapy is the first-line treatment for human ovarian cancer, chemoresistance remains a major obstacle to successful treatment, and there are currently no approved molecularly targeted therapies. Recent evidence indicates that signal transducer and activator of transcription-3 (STAT3) is a determinant of chemoresistance and is related to tumor recurrence in a large number of solid malignancies. In this study, we demonstrated that high levels of pSTAT3 were associated with chemoresistance in human ovarian cancer cells. Targeting STAT3 by siRNA technology markedly enhanced cisplatin-induced apoptosis in cisplatin-resistant ovarian cancer cells that expressed a high level of pSTAT3. Interleukin-6 (IL-6) could induce STAT3 activation in cisplatin-sensitive ovarian cancer cells and led to protection against cisplatin. The STAT3 siRNA treatment also blocked IL-6-induced STAT3 phosphorylation, resulting in the attenuation of the anti-apoptotic activity of IL-6. We found that the combination of cisplatin and STAT3 siRNA resulted in the collapse of the mitochondrial membrane potential, attenuated the expression of Bcl-xL and Bcl-2, and increased the release of cytochrome C and expression of Bax. Taken together, these results suggest that the pharmacological inhibition of STAT3 may be a promising therapeutic strategy for the management of chemoresistance in ovarian cancer.
Biochemical and Biophysical Research Communications 05/2013; 435(2). DOI:10.1016/j.bbrc.2013.04.087 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) have been considered to be the attractive vehicles for delivering therapeutic agents toward various tumor diseases. This study was to explore the distribution pattern, kinetic delivery of adenovirus, and therapeutic efficacy of the MSC loading of E1A mutant conditionally replicative adenovirus Adv-Stat3(-) which selectively replicated and expressed high levels of anti-sense Stat3 complementary DNA in breast cancer and melanoma cells.
We assessed the release ability of conditionally replicative adenovirus (CRAd) from MSC using crystal violet staining, TCID(50) assay, and quantitative PCR. In vitro killing competence of MSCs carrying Adv-Stat3(-) toward breast cancer and melanoma was performed using co-culture system of transwell plates. We examined tumor tropism of MSC by Prussian blue staining and immunofluorescence. In vivo killing competence of MSCs carrying Adv-Stat3(-) toward breast tumor was analyzed by comparison of tumor volumes and survival periods.
Adv-Stat3(-) amplified in MSCs and were released 4 days after infection. MSCs carrying Adv-Stat3(-) caused viral amplification, depletion of Stat3 and its downstream proteins, and led to significant apoptosis in breast cancer and melanoma cell lines. In vivo experiments confirmed the preferential localization of MSCs in the tumor periphery 24 hours after tail vein injection, and this localization was mainly detected in the tumor parenchyma after 72 hours. Intravenous injection of MSCs carrying Adv-Stat3(-) suppressed the Stat3 pathway, down-regulated Ki67 expression, and recruited CD11b-positive cells in the local tumor, inhibiting tumor growth and increasing the survival of tumor-bearing mice.
These results indicate that MSCs migrate to the tumor site in a time-dependent manner and could be an effective platform for the targeted delivery of CRAd and the amplification of tumor killing effects.
Molecular Cancer 11/2011; 10(1):134. DOI:10.1186/1476-4598-10-134 · 4.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: P21(WAF1/Cip1) binds to cyclin-dependent kinase complexes and inhibits their activities. It was originally described as an inhibitor of cancer cell proliferation. However, many recent studies have shown that p21 promotes tumor progression when accumulated in the cell cytoplasm. So far, little is known about the correlation between cytoplasmic p21 and drug resistance. This study was aimed to investigate the role of p21 in the cisplatin resistance of ovarian cancer.
RT-PCR, western blot and immunofluorescence were used to detect p21 expression and location in cisplatin-resistant ovarian cancer cell line C13* and its parental line OV2008. Regulation of cytoplasmic p21 was performed through transfection of p21 siRNA, Akt2 shRNA and Akt2 constitutively active vector in the two cell lines; their effects on cisplatin-induced apoptosis were evaluated by flow cytometry. Tumor tissue sections of clinical samples were analyzed by immunohistochemistry.
p21 predominantly localizes to the cytoplasm in C13* compared to OV2008. Persistent exposure to low dose cisplatin in OV2008 leads to p21 translocation from nuclear to cytoplasm, while it had not impact on p21 localization in C13*. Knockdown of cytoplasmic p21 by p21 siRNA transfection in C13* notably increased cisplatin-induced apoptosis through activation of caspase 3. Inhibition of p21 translocation into the cytoplasm by transfection of Akt2 shRNA into C13* cells significantly increased cisplatin-induced apoptosis, while induction of p21 translocation into the cytoplasm by transfection of constitutively active Akt2 in OV2008 enhanced the resistance to cisplatin. Immunohistochemical analysis of clinical ovarian tumor tissues demonstrated that cytoplasmic p21 was negatively correlated with the response to cisplatin based treatment.
Cytoplasmic p21 is a novel biomarker of cisplatin resistance and it may represent a potential therapeutic target for ovarian tumors that are refractory to conventional treatment.
BMC Cancer 09/2011; 11(1):399. DOI:10.1186/1471-2407-11-399 · 3.36 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cisplatin-centered chemotherapy is the first-line treatment for human ovarian cancer. However, chemoresistance remains a major obstacle to successful treatment. Evidence has indicated that signal transducer and activator of transcription-3 (STAT3) is a determinant of chemoresistance; it was related to tumor recurrence in a large number of solid malignancies. Unfortunately, none of the compounds currently developed to block STAT3 signaling has been considered a serious clinical candidate because of toxicity or limited bioavailability. In this study, we clarified the significance of STAT3 activation in chemoresistant ovarian cancer and assessed the suitability of a novel oncolytic adenovirus (M4) designed to specifically deplete STAT3 and reverse cisplatin resistance in ovarian cancer. We showed that aberrant expression and constitutive activation of STAT3 was instrumental in cisplatin resistance in ovarian cancer cell lines and in ovarian cancer tissue samples. The M4 adenovirus could specifically deplete constitutive and inducible STAT3 and phosphorylated STAT3 proteins in ovarian cancer cells. This significantly inhibited cell survival and enhanced cisplatin-induced apoptosis. In contrast, normal human umbilical vein endothelial cells and human ovarian surface epithelial cells appeared to be unaffected by M4 treatment. Furthermore, a combined cisplatin plus M4 therapy substantially eliminated populations enriched in tumor-initiating cells. In mice, systemic intraperitoneal administration of M4 significantly potentiated the antitumor effect of cisplatin. These results suggest that M4 has great potential as a therapy against cisplatin resistance in human ovarian cancer. Thus, it warrants further clinical investigation.
Human gene therapy 08/2011; 23(1):32-45. DOI:10.1089/hum.2011.101 · 3.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although dramatic clinical success has been achieved in acute promyelocytic leukemia (APL), the success of differentiating agents has not been reproduced in non-APL leukemia. A key barrier to the clinical success of arsenic is that it is not potent enough to achieve a clinical benefit at physiologically tolerable concentrations by targeting the leukemia cell differentiation pathway alone. We explored a novel combination approach to enhance the eradication of leukemia stem cells (LSCs) by arsenic in non-APL leukemia. In the present study, phosphatidylinositol 3-kinase /AKT/mammalian target of rapamycin (mTOR) phosphorylation was strengthened after As(2)S(2) exposure in leukemia cell lines and stem/progenitor cells, but not in cord blood mononuclear cells (CBMCs). propidium iodide-103, the dual PI3K/mTOR inhibitor, effectively inhibited the transient activation of the PI3K/AKT/mTOR pathway by As(2)S(2). The synergistic killing and differentiation induction effects on non-APL leukemia cells were examined both in vitro and in vivo. Eradication of non-APL LSCs was determined using the nonobese diabetic/severe combined immunodeficiency mouse model. We found that a combined As(2)S(2)/PI-103 treatment synergized strongly to kill non-APL leukemia cells and promote their differentiation in vitro. Furthermore, the combined As(2)S(2)/PI-103 treatment effectively reduced leukemia cell repopulation and eradicated non-APL LSCs partially via induction of differentiation while sparing normal hematopoietic stem cells. Taken together, these findings suggest that induction of the PI3K/AKT/mTOR pathway could provide a protective response to offset the antitumor efficacy of As(2)S(2). Targeting the PI3K/AKT/mTOR pathway in combination with As(2)S(2) could be exploited as a novel strategy to enhance the differentiation and killing of non-APL LSCs.
[Show abstract][Hide abstract] ABSTRACT: Human papillomavirus (HPV) E2 gene disruption is one of the key features of HPV-induced cervical malignant transformation. Though it is thought to prevent progression of carcinogenesis, the pro-apoptotic function of E2 protein remains poorly understood. This study shows that expression of HPV16 E2 induces apoptosis both in HPV-positive and -negative cervical cancer cell lines and leads to hyperactivation of caspase-8 and caspase-3. Activation of these signaling factors is responsible for the observed sensitivity to apoptosis upon treatment with anti-Fas antibody or TNF-α. In addition, immunoprecipitation experiments clearly show an interaction between HPV16 E2 and c-FLIP, a key regulator of apoptotic cell death mediated by death receptor signaling. Moreover, c-FLIP and a caspase-8 inhibitor protect cells from HPV16 E2-mediated apoptosis. Overexpression of c-FLIP rescues cervical cancer cells from apoptosis induced by HPV16 E2 protein expression. The data suggest that HPV16 E2 abrogates the apoptosis-inhibitory function of c-FLIP and renders the cell hypersensitive to the Fas/FasL apoptotic signal even below threshold concentration. This suggests a novel mechanism for deregulation of cervical epithelial cell growth upon HPV-induced transformation, which is of great significance in developing therapeutic strategies for intervention of cervical carcinogenesis.
[Show abstract][Hide abstract] ABSTRACT: Abnormal lymphangiogenesis is associated with several diseases such as tumor metastasis and lymphangioma. Human lymphangioma originated from the transformation of lymphatic endothelium is a benign malformation of lymphatic vessels and its pathogenesis has up to date not been illuminated and its cell model has also not been established. An optimized method was used to isolate lymphatic endothelial cells from human glossal lymphangioma (GL-LECs) and GL-LECs were further primarily cultured and expanded. GL-LECs were of typical cobblestone appearance when they reached confluence. The weible-palade body was observed in the GL-LECs cytoplasm. Almost all GL-LECs were strongly positive for specific lymphatic markers FLT-4, LYVE-1 and prox-1 by immunocytochemistry. Furthermore, three-dimension tube-like capillaries of GL-LECs resembled the lymphatic system in vivo, and the GL-LECs spheroids sprouted radically out to form three-dimensional buds when embedded in the cultured BME. These results indicated that high purity GL-LECs were successfully isolated and expanded. They had the abilities of tube formation and differentiation in vitro, which provide a favorable cell model for further uncovering the pathogenesis of human lymphangiomas.
[Show abstract][Hide abstract] ABSTRACT: Tumor cells acquire the ability to proliferate uncontrollably, resist apoptosis, sustain angiogenesis and evade immune surveillance. Signal transducer and activator of transcription (STAT) 3 regulates all of these processes in a surprisingly large number of human cancers. Consequently, the STAT3 protein is emerging as an ideal target for cancer therapy. This paper reports the generation of an oncolytic adenovirus (M4), which selectively blocks STAT3 signaling in tumor cells as a novel therapeutic strategy. M4 selectively replicated in tumor cells and expressed high levels of antisense STAT3 complementary DNA during the late phase of the viral infection in a replication-dependent manner. The viral progeny yield of M4 in tumor cells was much higher than that of the parent adenoviral mutants, Ad5/dE1A. M4 effectively silenced STAT3 and its target genes in tumor cells while sparing normal cells and exhibited potent antitumoral efficacy in vitro and in vivo. Systemic administration of M4 significantly inhibited tumor growth in an orthotopic gastric carcinoma mouse model, eliminated abdominal cavity metastases and prolonged survival time. In summary, M4 has low toxicity and great potential as a therapeutic agent for different types of cancers.
[Show abstract][Hide abstract] ABSTRACT: Histone deacetylase was overexpressed in a variety of cancers and was closely correlated with oncogenic factors. The histone deacetylase inhibitor, trichostatin A (TSA) was shown to induce apoptosis in many cancer cells. However, the mechanism of TSA on induction of cancer cells apoptosis is poorly understood. This study was designed to characterize the global gene expression profiles before and after treatment of human leukemia cell line Molt-4 with TSA. Flow cytometry, MTT and DNA ladder were used to observe the effect of TSA on the apoptosis of MOLT-4 cells and normal human peripheral blood mononuclear cells (PBMC). Microarray, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect the difference of gene and protein expressions of Molt-4 cells after incubation of the cells with TSA. The results showed that TSA could induce Molt-4 apoptosis in dose- and time-dependent manners but spared PBMCs. Microarray analysis showed that after incubation with TSA for 9 h, 310 genes were upregulated and 313 genes were deregulated. These genes regulate the growth, differentiation and survival of cells. Among these genes, STAT5A was down-regulated by 80.4% and MYC was down-regulated by 77.3%. It was concluded that TSA has definite growth-inhibiting and apoptosis-inducing effects on Molt-4 cells in time- and dose-dependent manners, with weak cytotoxic effects on PBMCs at the same time. The mechanism of TSA selectively inducing apoptosis and inhibiting growth may be ascribed to the changes of pro-proliferation genes and anti-apoptosis genes.
Journal of Huazhong University of Science and Technology 09/2009; 29(4):445-50. DOI:10.1007/s11596-009-0411-y · 0.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diverse types of voltage-gated potassium (K+) channels have been shown to be involved in regulation of cell proliferation. The maxi-conductance Ca2+-activated K+ channels (BK channels) may play an important role in the progression of human cancer. To explore the role of BK channels in regulation of apoptosis in human ovarian cancer cells, the effects of the specific BK channel activator NS1619 on induction of apoptosis in A2780 cells were observed. Following treatment with NS1619, cell proliferation was measured by MTT assay. Apoptosis of A2780 cells pretreated with NS1619 was detected by agarose gel electrophoresis of cellular DNA and flow cytometry. Our data demonstrate that NS1619 inhibits the proliferation of A2780 cells in a dosage and time dependent manner IC50=31.1 microM, for 48 h pretreatment and induces apoptosis. Western blot analyses showed that the anti-proliferation effect of NS1619 was associated with increased expression of p53, p21, and Bax. These results indicate that BK channels play an important role in regulating proliferation of human ovarian cancer cells and may induce apoptosis through induction of p21(Cip1) expression in a p53-dependent manner.
Biochemical and Biophysical Research Communications 11/2008; 375(2):205-9. DOI:10.1016/j.bbrc.2008.07.161 · 2.30 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Oncolytic adenoviruses represent a promising novel therapeutic option for the treatment of cancer. Despite their demonstrated safety in human clinical trials, the fundamental properties of oncolytic adenovirus biodistribution, spread, viral persistence, and replication in vivo have not been well characterized. The aim of this study was to evaluate the kinetics of viral distribution, spread, replication, and antitumoral efficacy after i.v. administration of a novel oncolytic mutant M1. This mutant consists of the E1A CR2-deleted Adv5 with a fragment of antisense polo-like kinase 1 (plk1) cDNA inserted into the deleted 6.7K/gp19K region, which combines oncolytic properties with efficient plk1 silencing, as described in our previous reports. In the present study, we established a new human orthotopic gastric carcinoma with a high frequency metastasis mouse model and showed that M1 spread not only in local primary tumors but also in disseminated metastases. M1 could effectively replicate in tumor cells leading to "oncolysis" and was able to eliminate expression of the targeted gene plk1 in human orthotopic gastric carcinoma model mice. Therefore, i.v. administration of M1 could prolong the survival time of tumor-bearing mice.
Molecular Cancer Therapeutics 07/2008; 7(6):1624-32. DOI:10.1158/1535-7163.MCT-07-2134 · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ezrin primarily acts as a linker between the plasma membrane and the cytoskeleton and is a key component in tumor metastasis. In the present study, RNA interference (RNAi) using ezrin small hairpin RNAs (ezrin shRNAs) was used to define the roles of ezrin in the regulation of malignant behaviors of human breast cancer. The highly metastatic human breast cancer cell MDA-MB-231, in which ezrin mRNA and protein levels are the highest, was selected as a cell model in vitro. In addition, we also found that ezrin expression was up-regulated and its immuno-staining trans-located from cell membrane to cytoplasm, whereas E-cadherin expression decreased and showed the same cell distribution as ezrin in lymphatic metastases of human breast carcinomas. After repression of ezrin by more than 85% of G3PDH and 75% of beta-actin in mRNA and protein levels was maintained in the stable expressing ezrin shRNAs MDA-MB-231 cell clones, the abilities of cell motility and invasiveness were obviously inhibited with a 4-fold and 2-fold, respectively, and the altered cell polarity was observed. Western blot analyses further revealed that the silencing of ezrin induced an increased E-cadherin expression and a decreased phosphorylation of beta-catenin by inhibiting phosphorylation levels of c-src. These data indicate that ezrin overexpression positively correlated with metastatic potentials of human breast cancer cells, especially lymphatic system metastasis. Decreased ezrin expression by shRNA reversed metastatic behaviors of human breast cancer cells by inducing c-src-mediated E-cadherin expression, suggesting that ezrin may have potential values in assessing lymphatic metastasis of human breast cancers.
Cancer Letters 04/2008; 261(1):55-63. DOI:10.1016/j.canlet.2007.11.018 · 5.62 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A significant proportion of prostate cancer patients treated with curative intent develop advanced disease. At a fundamental biological level, very little is known about what makes the disease aggressive and metastatic. Observational pathology reports and experimental data suggest that an epithelial-mesenchymal transition (EMT) is involved in prostate cancer invasiveness. The mechanism by which vimentin promotes prostate cancer cell invasion and metastasis was examined. The highly metastatic human prostate epithelial cell line PC-3M-1E8 (1E8-H) and the low metastatic line PC-3M-2B4 (2B4-L) were used for comparative proteomic analysis by two-dimensional gel electrophoresis, followed by matrix-assisted laser desorption/time of flight mass spectrometry (MALDI-TOF-MS). A transwell assay was performed to test cell migration and invasion and immunoblotting was used to analyze the relative expression of proteins. High vimentin expression was detected in 1E8-H compared to 2B4-L cells. Down-regulation of vimentin in 1E8-H by antisense-vimentin transfection led to a significant inhibition of invasiveness, and selective stimulation of vimentin activity in 2B4-L by delivery of recombinant vimentin promoted cell invasiveness. Vimentin activity was associated with C-src, beta-catenin and E-cadherin expression. PP2, a specific inhibitor of src family kinases, reduced phospho-beta-catenin expression and induce E-cadherin expression. Vimentin promotes tumor cell invasiveness and the targeting of vimentin/C-src may be a promising strategy for preventing or blocking prostate cancer metastasis.
Anticancer research 01/2008; 28(1A):327-34. · 1.83 Impact Factor