In vitro and in vivo anticancer efficacy of silibinin against human pancreatic cancer BxPC-3 and PANC-1 cells

Silibinin suppresses the growth of many cancers; however, its efficacy against pancreatic cancer has not been evaluated in established preclinical models. Here, we investigated in vitro and in vivo effects of silibinin against lower and advanced stages of human pancreatic carcinoma cells. Silibinin (25–100 μM) treatment for 24–72 h caused a dose- and time-dependent cell growth inhibition of 27–77% (P < 0.05–0.001) in BxPC-3 cells, and 22–45% (P < 0.01–0.001) in PANC-1 cells. Silibinin showed a strong dose-dependent G1 arrest in BxPC-3 cells (upto 72% versus 45% in control; P < 0.001), but a moderate response in advanced PANC-1 cells. Cell death observed in cell growth assay, was accompanied by up to 3-fold increase (P < 0.001) in apoptosis in BxPC-3 cells, and showed only slight effect on PANC-1 cells. Dietary feeding of silibinin (0.5%, w/w in AIN-93M diet for 7 weeks) inhibited BxPC-3 and PANC-1 tumor xenografts growth in nude mice without any apparent change in body weight gain and diet consumption. Tumor volume and weight were decreased by 47% and 34% (P ? 0.001) in BxPC-3 xenograft, respectively. PANC-1 xenograft showed slower growth kinetics and silibinin decreased tumor volume by 34% (P < 0.001) by 7 weeks. Another 4 weeks of silibinin treatment to PANC-1 xenograft showed 28% and 33% decrease in tumor volume and weight, respectively. Silibinin-fed group of BxPC-3 tumors showed decreased cell proliferation and angiogenesis and an increased apoptosis, however, considerable inhibitory effect was observed only for angiogenesis in PANC-1 tumors. Overall, these findings show both in vitro as well as in vivo anticancer efficacy of silibinin against pancreatic cancer that could involve inhibition of cell proliferation, cell cycle arrest, apoptosis induction and/or decrease in tumor angiogenesis.     

Carboxymethyl benzylamide dextran inhibits angiogenesis and growth of VEGF-overexpressing human epidermoid carcinoma xenograft in nude mice

Vascular endothelial growth factor (VEGF) expression is elevated in a wide variety of solid tumours. Inhibition of VEGF activities is able to reduce angiogenesis and tumour growth. We have recently shown in vitro that carboxymethyl dextran benzylamide (CMDB7) prevents the binding of VEGF(165) to its cell surface receptors and thus inhibits VEGF activities on endothelial cells. In the present study, we explored the effects of CMDB7 on highly aggressive human epidermoid carcinoma A431 cells known to overexpress epidermal growth factor receptors (EGFRs) and produce a high amount of VEGF and a minor quantity of bFGF. In vitro, CMDB7 blocked the mitogenic activity of A431-conditioned medium on endothelial cells. Concerning A431 cells, CMDB7 inhibited their proliferation and the VEGF(165) binding to them. In vivo, administration of CMDB7 (10 mg kg(-1)) three times per week for 2 weeks inhibited the growth of A431 xenografts in nude mice by 73% as compared to the control group. Immunostaining of endothelial cells with mouse-specific GSL-1 lectin in tumour sections revealed that CMDB7 also inhibited the density of intratumour endothelial cells by 66%. These findings demonstrate that CMDB7 has an efficient antiangiogenic and antitumour action in vivo even when tumour cells produce a high level of VEGF and EGFRs.     

Dkk-1 secreted by mesenchymal stem cells inhibits growth of breast cancer cells via depression of Wnt signalling

We determined the molecular mechanism of inhibitory effect of human mesenchymal stem cells (hMSCs) on the growth of human MCF-7 breast cancer cells. Our finding showed that beta-catenin was down-regulated in MCF-7 cells by conditioned media from Z3 hMSCs, and the expression level of dickkopf-1 (Dkk-1) was higher in Z3 cells than that in MCF-7 cells. Neutralization of Dkk-1 and small interference RNA targeting Dkk-1 mRNA in Z3 cells attenuated the inhibitory effect of Z3 cells on MCF-7 cells. Overexpression of Dkk-1 in Z3 cells enhanced the inhibition. Therefore, Dkk-1 secreted by Z3 cells involves the inhibition via the Wnt pathway.     

Diosgenin,a steroidal saponin,inhibits STAT3 signaling pathway leading to suppression of proliferation and chemosensitization of human hepatocellular carcinoma cells

Constitutive activation of STAT3 has been shown in several human cancers and transformed cell lines including hepatocellular carcinoma (HCC). In the present report, we investigated whether diosgenin, a steroidal saponin isolated from fenugreek can modulate the STAT3 signaling pathway. We found that diosgenin inhibited both constitutive and inducible activation of STAT3 with no effect on STAT5. The activation of c-Src, JAK1 and JAK2 implicated in STAT3 activation, were also suppressed by this saponin. Pervanadate reversed the diosgenin-induced downregulation of STAT3, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that diosgenin can induce the expression of Src homology 2 phosphatase 2 (SH-PTP2) that correlated with downregulation of constitutive STAT3 activation. Diosgenin also downregulated the expression of various STAT3-regulated gene products, inhibited proliferation and potentiated the apoptotic effects of paclitaxel and doxorubicin. Overall, these results suggest that diosgenin is a novel blocker of the STAT3 activation pathway, with a potential role in the treatment of HCC and other cancers.     

Enhanced anti-tumor activity by the combination of TRAIL/Apo-2L and combretastatin A-4 against human colon cancer cells via induction of apoptosis in vitro and in vivo

The present study indicated that the combination of TRAIL/Apo-2L and CA-4 exerted synergistic anti-proliferative effect against human colon carcinoma cells including SW-620 and HCT-116 in vitro. Moreover, the increased anti-tumor efficacy of TRAIL/Apo-2L combined with CA-4 was further validated on SW-620 xenograft model in nude mice. These enhanced anti-cancer activities were accompanied by caspase-mediated apoptosis. Furthermore, it was identified that NF-κB as the major determinant of TRAIL/Apo-2L resistance could be blocked in cytoplasm by TRAIL/Apo-2L plus CA-4 treatment. Taken together, these findings build the rationale for further (pre)clinical development of TRAIL/Apo-2L and CA-4 against colorectal cancer.     

扁桃体鳞癌中HPV感染与EGFR、VEGF表达的相关性研究

目的:研究扁桃体鳞癌(squamous cell carcinomas of the tonsil tonsillar,SCCs)中人乳头瘤病毒(human papillomavirus,HPV)感染与血管内皮生长因子(vascular endothelial growth factor,VEGF)和表皮生长因子受体(epidermal growth factor,EGFR)表达的关系,探讨HPV与EGFR和VEGF在扁桃体鳞癌发生中的交互效应。方法:采用多重实时荧光定量聚合酶链反应(multiplex real-time polymerase chain reaction,MT-PCR)检测85例扁桃体鳞癌HPV DNA及型别,通过HPV DNA分析进行HPV感染状况的测定;同时采用半定量免疫组化检测HPV(+)组和HPV(-)组中VEGF、EGFR蛋白表达情况。结果:扁桃体鳞癌中HPV感染率为49.4%(42/85),HPV-16型占所有感染者的比例为90.5%(38/42),明显高于其他型别;HPV(+)组EGFR蛋白表达明显低于HPV(-)组(P值均<0.01);HPV(+)组与HPV(-)组VEGF蛋白表达无明显差异;此外,VEGF表达与EGFR、患者的年龄、性别、TNM分期、组织学分级均无明显相关性。结论:HPV感染与扁桃体鳞癌的发生存在相关性,HPV相关的扁桃体鳞癌中,HPV基因可能通过改变EGFR表达致癌,为HPV致癌机理进一步研究提供一个新的视角,具有重要的理论意义。     

Reduction of PTEN protein and loss of epidermal growth factor receptor gene mutation in lung cancer with natural resistance to gefitinib (IRESSA)

Gefitinib (IRESSA), an epidermal growth factor receptor (EGFR) tyrosine kinase (TK) inhibitor, has antitumour activity in the advanced non-small-cell lung cancer (NSCLC) setting. However, in chemotherapy-naïve patients with advanced NSCLC, the addition of gefitinib to standard chemotherapy regimens failed to increase survival. These results suggest the need for improved patient selection and combination rationales for targeted therapies. We have identified subpopulations of an adenocarcinoma cell line that are naturally resistant to gefitinib, and have analysed the cDNA expression profiles, genomic status of EGFR gene and the effect of gefitinib on signalling pathways in these cell lines in order to identify key mechanisms for naturally acquired resistance to gefitinib. Gefitinib-resistant subpopulations demonstrated increased Akt phosphorylation (not inhibited by gefitinib), reduced PTEN protein expression and loss of the EGFR gene mutation when compared with parental cell lines. These differences in Akt and PTEN protein expression were not evident from the cDNA array profiles. These data suggests that (1) the EGFR gene mutation may be possibly lost in some cancer cells with other additional mechanisms for activating Akt, (2) reintroduction of PTEN or pharmacological downregulation of the constitutive PI3K–Akt-pathway activity may be an attractive therapeutic strategy in cancers with gefitinib resistance.     

Transcription factor KLF9 suppresses the growth of hepatocellular carcinoma cells in vivo and positively regulates p53 expression

Krüppel-like factor 9 (KLF9) is known to be a tumor suppressor gene in colorectal tumors and glioblastoma; however, the functional status and significance of KLF9 in hepatocellular carcinoma (HCC) is unclear. We report here that KLF9 is downregulated in HCC tissues. Restoration of KLF9 significantly inhibited growth and caused apoptosis in SK-Hep1 and HepG2 cells. We found that KLF9 positively regulated p53 levels by directly binding to GC boxes within the proximal region of the p53 promoter. Moreover, in the presence of cycloheximide, KLF9 significantly increased p53 stability in HCC cells. Remarkably, ectopic expression of KLF9 was sufficient to delay the onset of tumors and to promote regression of the established tumors in vivo, suggesting that KLF9 plays a critical role in HCC development and that pharmacological or genetic activation of KLF9 may have potential in the treatment of HCC.     

Dual inhibition of epidermal growth factor and insulin-like 1 growth factor receptors reduce intestinal adenoma burden in the Apc(min/+) mouse

Background:

Identification of early molecular pathway changes may be useful as biomarkers for tumour response/resistance prediction, and here we provide direct in vivo proof of this concept. The type 1 insulin-like growth factor receptor (IGF1R) has been implicated in various aspects of adenoma development and metastasis. We show here that, in murine intestinal adenomas acutely exposed to a small molecular inhibitor of EGFR (gefitinib), there is concurrent suppression of EGFR downstream signalling and induction of IGF signalling. We therefore tested the hypothesis that blockade of EGFR signalling was being tempered by compensatory activation of the IGF pathway by examining the effect of chronic suppression of IGF1R using AZ12253801, a small molecular tyrosine kinase inhibitor of IGF1R.

Methods:

Male Apc^min/+ mice with an intestinal tumour burden were exposed to a single dose of an inhibitor against EGFR (gefitinib), IGF1R (AZ12253801), 0.5% Tween 80 or combined EGFR/IGF1R inhibitor and culled 4 h post dosing. Tumour tissue was analysed to detect the early molecular pathways induced and anti-tumour phenotypic changes. Cohorts of male Apc^min/+ mice (n=15–17) were subsequently treated with gefitinib for a period of 8 weeks and subsequently exposed to single (either gefitinib or AZ12253801) or combined (gefitinib and AZ12253801) therapy. We also included a vehicle-treated cohort, which was never exposed to gefitinib and became symptomatic of the disease by day 150.

Results:

Both single treatments delayed the onset of disease symptoms. Combined dosing with gefitinib and AZ12253801 similarly delayed the onset of symptoms, and at 200 days suppressed small intestinal tumourigenesis more effectively than either treatment alone (median small intestinal adenoma volume (47 mm³ (comb) vs 248 mm³ (AZ12253801), P=0.0003 and 47 mm³ (comb) vs 123 mm³ (gefitinib), P=0.0042, Mann–Whitney (two-sided) test).

Conclusion:

Our data provide evidence in support of the use of combinatorial therapy, and establishes the need to further define the precise benefit in vivo.     

EGCG inhibits activation of the insulin-like growth factor (IGF)/IGF-1 receptor axis in human hepatocellular carcinoma cells

The receptor tyrosine kinase (RTK) insulin like growth factor-1 (IGF-1)/IGF-1 receptor (IGF-1R) axis plays an important role in the development of hepatocellular carcinoma (HCC). EGCG inhibits activation of the various types of RTKs and that this is associated with inhibition of multiple downstream signaling pathways. In this study we examined the effects of EGCG on activity of the IGF/IGF-1R axis in HepG2 human HCC cells which express constitutive activation of this axis. The level of phosphorylated (i.e. activated) form of the IGF-1R protein (p-IGF-1R) was increased in a series of human HCC cell lines when compared with the Hc normal human hepatocytes. EGCG preferentially inhibited growth of HepG2 cells when compared with Hc cells. Treatment of HepG2 cells with EGCG induced apoptosis and caused a decrease in the p-IGF-1R protein and its downstream signaling molecules including the p-ERK, p-Akt, p-Stat-3, and p-GSK-3β proteins, both in the absence or presence of ligand stimulation. EGCG also decreased the levels of both IGF-1 and IGF-2 proteins and mRNAs, but increased the levels of the IGFBP-3 protein. These findings suggest that EGCG can overcome the stimulatory effects of IGFs on the IGF-1R dependent signaling pathway, thus expanding the roles of EGCG as an inhibitor of critical RTKs involved in HCC cell proliferation. These results provide further evidence that EGCG may be useful in the chemoprevention or treatment of liver cancer.     

Anesthetic pentobarbital inhibits proliferation and migration of malignant glioma cells

Malignant gliomas are common and aggressive brain tumors in adults. The rapid proliferation and diffuse brain migration are main obstacles to successful treatment. Here we show that pentobarbital, a central depressant introduced clinically a century ago, is capable of suppressing proliferation and migration of C6 malignant glioma cells in a concentration-dependent manner. Pentobarbital also leads to a G1 phase cell cycle arrest accompanied by suppressed G1 cell cycle regulatory proteins Cyclin D1, Cyclin D3, CDK2 and phosphorylated Rb. In addition, noticeable morphological changes and interrupted α-tubulin microtubule assembly are induced by pentobarbital exposure. Intracellular signal pathways involved in the effect of pentobarbital is concerned with inactivation of ERK, c-Jun and Akt. Together, these findings suggest anti-proliferation and anti-migration effects of pentobarbital on malignant gliomas, most likely by arresting cell cycle and interfering microtubule. ERK, c-Jun MAPK and PI3K/Akt are possible signaling pathways involved.     

Magnetic resonance imaging in an orthotopic rat model: blockade of epidermal growth factor receptor with EMD72000 inhibits human pancreatic carcinoma growth

The purpose of our research was to investigate the antiangiogenic effect of the epidermal growth factor receptor monoclonal antibody (anti-EGF-R MAB) EMD72000, in an orthotopic human pancreatic carcinoma model in rats, assessed by magnetic resonance (MR) imaging using angiogenic surrogate markers in comparison with histopathologic findings. Human pancreatic adenocarcinoma cells L3.6pl were injected orthotopically in the pancreas of 12 athymic nude rats. Through a 21-day course, groups of 6 rats were treated intraperitoneally with either EMD72000 or with saline solution for control animals. Dynamic contrast-enhanced MR imaging was performed before and after the treatment to assess microvascular permeability, estimated by the endothelial transfer coefficient (KPS) and fractional plasma volumes (fPV) of the pancreatic tumors. EMD72000-treated animals showed significantly less tumor volume progression (1,080 mm3 +/- 1,244; p = 0.012) and significantly lower values for microvascular permeability (KPS = 4.2 ml min(-1) 100 ml(-1) of tissue +/- 2.8; p = 0.015), fractional plasma volume (fPV = 0.018 ml ml(-1) of tissue +/- .015; p = 0.003) and microvessel density (MVD = 13 +/- 4 (0.159 mm2); p = 0.001) than saline-treated animals (6,544 mm3 +/- 5,202; 9.5 ml min(-1) 100 ml(-1) of tissue +/- 4.3, 0.056 ml ml(-1) of tissue +/- 0.019 and 25 +/- 5 (0.159 mm2), respectively). KPS and fPV values showed moderate positive correlation with MVD (r = 0.5, p = 0.103; r = 0.6, p = 0.065, respectively). Intraperitoneal injection of EMD72000 inhibits orthotopic human pancreatic carcinoma growth in rats. Antiangiogenic effects of anti-EGF-R MAB EMD72000 can be quantified and monitored noninvasively by dynamic MR imaging.     

Inhibition of tumor cell migration and metastasis by the proton-sensing GPR4 receptor

GPR4 is a member of the proton-sensing G protein-coupled receptor family. Within tumor microenvironments, the interstitial acidic pH may activate GPR4 to regulate the behavior of tumor cells. Mouse B16F10 melanoma cells and TRAMP-C1 prostate cancer cells, genetically engineered to overexpress GPR4 or the control vector, were subject to a series of cell migration, invasion and metastasis assays. Upon GPR4 overexpression and activation in an acidic pH, the migration of B16F10 and TRAMP-C1 cells was substantially inhibited in comparison to the vector control. Similar results were observed in the Matrigel invasion and transendothelial invasion assays. At the molecular level, stimulation of GPR4 by acidosis induced the activation of RhoA and the formation of actin stress fibers. In addition, treating B16F10 cells with the known Rho activator CN01 (calpeptin) strongly inhibited cell migration, recapitulating the acidosis/GPR4-induced motility inhibition phenotype. To examine the biological effects in vivo, B16F10 melanoma cells were intravenously injected into syngeneic C57BL/6 mice and pulmonary metastasis was inhibited by approximately 80% in GPR4-overexpressing B16F10 cells in comparison to the vector control. Upon treatment with the Rho activator CN01, the phenotype of the B16F10 vector cells paralleled that of the GPR4-overexpressing cells in cell migration and metastasis assays. These findings suggest that GPR4 activation by an acidic pH inhibits tumor cell migration and invasion, and the Rho GTPase is at least partly responsible for this phenotype.     

Dasatinib blocks cetuximab- and radiation-induced nuclear translocation of the epidermal growth factor receptor in head and neck squamous cell carcinoma

Background and purpose

The aberrant expression of epidermal growth factor receptor (EGFR) has been linked to the etiology of head and neck squamous cell carcinoma (HNSCC). The first major phase III trial combining cetuximab with radiation confirmed a strong survival advantage. However, both cetuximab and radiation can promote EGFR translocation to the nucleus where it enhances resistance to both of these modalities. In this report we sought to determine how to block cetuximab- and radiation-induced translocation of EGFR to the nucleus in HNSCC cell lines.

Material and methods

We utilized three established HNSCC cell lines, SCC1, SCC6 and SCC1483 and measured nuclear translocation of EGFR after treatment with cetuximab or radiation. We then utilized dasatinib (BMS-354825), a potent, orally bioavailable inhibitor of several tyrosine kinases, including the Src family kinases, to determine if SFKs blockade could abrogate cetuximab- and radiation-induced nuclear EGFR translocation.

Results

Cetuximab and radiation treatment of all three HNSCC lines lead to translocation of the EGFR to the nucleus. Blockade of SFKs abrogated cetuximab- and radiation-induced EGFR translocation to the nucleus.

Conclusions

The data presented in this report suggest that both cetuximab and radiation can promote EGFR translocation to the nucleus and dasatinib can inhibit this process. Collectively these findings may suggest that dasatinib can limit EGFR translocation to the nucleus and may enhance radiotherapy plus cetuximab in HNSCC.     

VEGF及其受体KDR在非小细胞肺癌中的表达及与肿瘤血管形成关系

目的：探讨血管内皮生长因子（VEGF）及其受体（KDR）的表达与非小细胞肺癌（NSCLC）血管形成的关系。方法：分别应用免疫组织化学S-P法和原位分子杂交法检测62例NSCLC组织和16例肺的良性瘤样病变组织中的VEGF和KDR mRNA的表达，并对血管进行染色、计数。结果：62例肺癌组织中46例有VEGF的表达（占74．29％），阳性表达位于肿瘤细胞胞膜及胞质；KDR mRNA在49例中有阳性表达（占79．03％），阳性表达位于肿瘤血管内皮细胞、肿瘤细胞胞膜及胞质。VEGF和KDR mRNA的表达与有无淋巴结转移和临床病理分期密切相关；VEGF和KDR mRNA阳性表达组微血管密度明显高于阴性表达组，P〈0．01。结论：VEGF和KDR的表达与NSCLC的发生、发展和转移可能密切相关，可作为评估NSCLC患者预后的一项指标。     

Good response to pemetrexed in patients of lung adenocarcinoma with epidermal growth factor receptor (EGFR) mutations

There are no reports about epidermal growth factor receptor (EGFR) mutations and the responsiveness to pemetrexed treatment. In order to understand the influence of EGFR mutations on pemetrexed response, we performed EGFR sequencing of lung adenocarcinoma from patients undergoing pemetrexed treatment and analyzed their response to pemetrexed. The pemetrexed-treated lung adenocarcinoma patients with adequate specimens for EGFR sequencing and measurable target lesions were enrolled for response analysis. Demographic data, EGFR mutation status, response to pemetrexed, and survival data were collected. From January 2004 to December 2008, 156 patients with measurable target lesions and EGFR sequencing results had complete clinical data for analysis. The patients with EGFR mutations (N = 93) had a better response rate (p = 0.016) and longer progression-free survival (PFS) (p = 0.030) than those with wild type EGFR (N = 63). By multivariate analysis, those who had better performance status (p = 0.013) and EGFR mutations (p = 0.021) had a longer PFS. In conclusion, lung adenocarcinoma patients receiving pemetrexed with EGFR mutations have a better response rate and longer PFS than those with wild type EGFR.