Xenografts In Athymic Nude Mice Research Articles

CD146-targeted immunoPET and NIRF Imaging of Hepatocellular Carcinoma with a Dual-Labeled Monoclonal Antibody

Overexpression of CD146 has been correlated with aggressiveness, recurrence rate, and poor overall survival in hepatocellular carcinoma (HCC) patients. In this study, we set out to develop a CD146-targeting probe for high-contrast noninvasive in vivo positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging of HCCs. YY146, an anti-CD146 monoclonal antibody, was employed as a targeting molecule to which we conjugated the zwitterionic near-infrared fluorescence (NIRF) dye ZW800-1 and the chelator deferoxamine (Df). This enabled labeling of Df-YY146-ZW800 with 89Zr and its subsequent detection using PET and NIRF imaging, all without compromising antibody binding properties. Two HCC cell lines expressing high (HepG2) and low (Huh7) levels of CD146 were employed to generate subcutaneous (s.c.) and orthotopic xenografts in athymic nude mice. Sequential PET and NIRF imaging performed after intravenous injection of 89Zr-Df-YY146-ZW800 into tumor-bearing mice unveiled prominent and persistent uptake of the tracer in HepG2 tumors that peaked at 31.65 ± 7.15 percentage of injected dose per gram (%ID/g; n=4) 72 h post-injection. Owing to such marked accumulation, tumor delineation was successful by both PET and NIRF, which facilitated the fluorescence image-guided resection of orthotopic HepG2 tumors, despite the relatively high liver background. CD146-negative Huh7 and CD146-blocked HepG2 tumors exhibited significantly lower 89Zr-Df-YY146-ZW800 accretion (6.1 ± 0.5 and 8.1 ± 1.0 %ID/g at 72 h p.i., respectively; n=4), demonstrating the CD146-specificity of the tracer in vivo. Ex vivo biodistribution and immunofluorescent staining corroborated the accuracy of the imaging data and correlated tracer uptake with in situ CD146 expression. Overall, 89Zr-Df-YY146-ZW800 showed excellent properties as a PET/NIRF imaging agent, including high in vivo affinity and specificity for CD146-expressing HCC. CD146-targeted molecular imaging using dual-labeled YY146 has great potential for early detection, prognostication, and image-guided surgical resection of liver malignancies.

Recombinant vaccinia virus GLV-1h68 is a promising oncolytic vector in the treatment of cholangiocarcinoma.

Although early stage cholangiocarcinoma (CC) can be cured by surgical extirpation, the options for treatment of advanced stage CC are very few and suboptimal. Oncolytic virotherapy using replication-competent vaccinia virus (VACV) is a promising new strategy to treat human cancers. The ability of oncolytic VACV GLV-1h68 to infect, replicate in, and lyse three human CC cell lines was assayed in vitro and in subcutaneous flank xenografts in athymic nude mice. In this study, we have demonstrated that GLV-1h68 effectively infects and lyses three CC cell lines (KMC-1, KMBC, and KMCH-1) in vitro. Expression of the viral marker gene ruc-gfp facilitated real-time monitoring of infection and replication. Furthermore in athymic nude mice, a single dose of GLV-1h68 significantly suppressed tumor growth. The treatment was well tolerated in all animals. Recombinant VACV GLV-1h68 has significant oncolytic ability against CC both in vitro and in vivo. GLV-1h68 has the potential to be used clinically as a therapeutic agent against CC.

Coffee inhibits nuclear factor-kappa B in prostate cancer cells and xenografts

Chronic inflammation contributes to prostate cancer and the transcription factor Nuclear Factor-kappa B (NF-κB) is constitutively active in most such cancers. We examine the effects of coffee on NF-κB and on the regulation of selected genes in human-derived prostate cancer cells (PC3) and in PC3 xenografts in athymic nude mice. PC3 cells stably transduced with an NF-κB-luciferase reporter were used both in vitro and for xenografts. NF-κB activity was measured by reporter assays, DNA binding and in vivo imaging. Gene expression was measured in PC3 cells, xenografts and tumor microenvironment by low-density arrays. Western blotting of activated caspases was used to quantify apoptosis.Coffee inhibited TNFα-induced NF-κB activity and DNA-binding in PC3 cells. Furthermore, coffee increased apoptosis and modulated expression of a number of inflammation- and cancer-related genes in TNFα-treated PC3 cells. In vivo imaging revealed a 31% lower NF-κB-luciferase activation in the xenografts of the mice receiving 5% coffee compared to control mice. Interestingly, we observed major changes in gene expression in the PC3 cells in xenografts as compared to PC3 cells in vitro. In PC3 xenografts, genes related to inflammation, apoptosis and cytoprotection were down-regulated in mice receiving coffee, and coffee also affected the gene expression in the xenograft microenvironment. Our data demonstrate that coffee inhibits NF-κB activity in PC3 cells in vitro and in xenografts. Furthermore, coffee modulates transcription of genes related to prostate cancer and inflammation. Our results are the first to suggest mechanistic links between coffee consumption and prostate cancer in an experimental mouse model.

PET Imaging of Dll4 Expression in Glioblastoma and Colorectal Cancer Xenografts Using (64)Cu-Labeled Monoclonal Antibody 61B.

Delta-like ligand 4 (Dll4) expressed in tumor cells plays a key role to promote tumor growth of numerous cancer types. Based on a novel antihuman Dll4 monoclonal antibody (61B), we developed a (64)Cu-labeled probe for positron emission tomography (PET) imaging of tumor Dll4 expression. In this study, 61B was conjugated with the (64)Cu-chelator DOTA through lysine on the antibody. Human IgG (hIgG)-DOTA, which did not bind to Dll4, was also prepared as a control. The Dll4 binding activity of the probes was evaluated through the bead-based binding assay with Dll4-alkaline phosphatase. The resulting PET probes were evaluated in U87MG glioblastoma and HT29 colorectal cancer xenografts in athymic nude mice. Our results demonstrated that the 61B-DOTA retained (77.2 ± 3.7) % Dll4 binding activity of the unmodified 61B, which is significantly higher than that of hIgG-DOTA (0.06 ± 0.03) %. Confocal microscopy analysis confirmed that 61B-Cy5.5, but not IgG-Cy5.5, predominantly located within the U87MG and HT29 cells cytoplasm. U87MG cells showed higher 61B-Cy5.5 binding as compared to HT29 cells. In U87MG xenografts, 61B-DOTA-(64)Cu demonstrated remarkable tumor accumulation (10.5 ± 1.7 and 10.2 ± 1.2%ID/g at 24 and 48 h postinjection, respectively). In HT29 xenografts, tumor accumulation of 61B-DOTA-(64)Cu was significantly lower than that of U87MG (7.3 ± 1.3 and 6.6 ± 1.3%ID/g at 24 and 48 h postinjection, respectively). The tumor accumulation of 61B-DOTA-(64)Cu was significantly higher than that of hIgG-DOTA-(64)Cu in both xenografts models. Immunofluorescence staining of the tumor tissues further confirmed that tumor accumulation of 61B-Cy5.5 was correlated well with in vivo PET imaging data using 61B-DOTA-(64)Cu. In conclusion, 61B-DOTA-(64)Cu PET probe was successfully synthesized and demonstrated prominent tumor uptake by targeting Dll4. 61B-DOTA-(64)Cu has great potential to be used for noninvasive Dll4 imaging, which could be valuable for tumor detection, Dll4 expression level evaluation, and Dll4-based treatment monitoring.

Abstract 1453: Methods for developing patient derived xenografts in athymic nude mice

Abstract Patient derived xenografts (PDX) mouse models for many types of cancer have been developed recapitulating therapeutic responses in the patient for which the PDX model was derived. Essentially, live surgically resected or biopsy tumor tissues are directly implanted into immune incompetent mice to develop in vivo models to test therapies, follow tumor progression (gene mutations that occur with patient tumor progression do so in mice) or to better understand drug resistance. However, the use of PDX models also has its limitations and challenges. Our laboratory has attempted to develop PDX models in athymic nude mice for numerous cancer histotypes such as bladder, brain, breast, colon, kidney, lung, melanoma, ovarian, pancreas, sarcoma and thyroid. While melanoma and colon cancers have very high tumor take rates (73 - 100%), the other cancer histotypes do not (14 - 50%). We share our experience for each cancer histotype and the techniques used to enhance the success rates in athymic nude mice. We also address the other challenges that include expansion of tumors after cryopreservation and conversion of human tumors to mouse tumors and spontaneous lymphomas. We provide methods to test for human versus mouse content that include QPCR and immunohistochemistry Citation Format: Laura A. Marlow, James L. Miller, Brandy Edenfield, Adam C. Mathias, Louis K. Dawson, William F. Durham, Robert J. Mullin, Daniel L. Small, Aidan J. Synnott, Kevin Wu, John A. Copland. Methods for developing patient derived xenografts in athymic nude mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1453. doi:10.1158/1538-7445.AM2015-1453

Abstract 5388: Methionine S-adenosyltransferase 2A (MAT2A) inhibitors for cancer treatment

Abstract Methionine S-adenosyltransferase 2A (MAT2A) is the catalytic subunit for synthesis of S-adenosylmethionine (SAM), the principal methyl donor in many biological processes. MAT2A is up-regulated in many cancers, including liver cancer and colorectal cancer (CRC) and is a potentially important drug target. In normal liver, methionine S-adenosyltransferase 1 (MAT1), but not MAT2A is expressed. MAT2A is induced in liver cancer. We developed a family of fluorinated N,N-dialkylaminostilbene agents, called FIDAS agents, that inhibit the proliferation of CRC cells and liver cells in vitro and in vivo. Using a biotinylated FIDAS analog, we identified the catalytic subunit of MAT2A as the direct and exclusive binding target of these FIDAS agents. FIDAS agents do not bind MAT1A. MAT2B, an associated regulatory subunit of MAT2A, binds indirectly to FIDAS agents through its association with MAT2A. FIDAS agents inhibited MAT2A activity in SAM synthesis, and depletion of MAT2A by shRNAs inhibited CRC and liver cancer cell growth. FIDAS agents also inhibited specific histone methylation and repressed transcription of c-myc and other oncogenes. FIDAS agents delivered orally repressed CRC xenografts in athymic nude mice. Based on SAR and docking studies, new generations of FIDAS agents were synthesized and tested. An active in vivo metabolite of FIDAS was identified. PK/PD studies were performed. Our findings suggest that FIDAS analogs targeting MAT2A represent a family of novel and potentially useful agents for cancer treatment. Citation Format: Wen Zhang, Vitaliy Sviripa, David Watt, Chunming Liu. Methionine S-adenosyltransferase 2A (MAT2A) inhibitors for cancer treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5388. doi:10.1158/1538-7445.AM2015-5388

Abstract 1387: Enhanced recruitment of tumor microenvironment components in A549 xenografts with acquired resistance to sorafenib

Abstract Acquired drug resistance represents a major obstacle to effective cancer treatment. Sorafenib is an oral multikinase inhibitor with proven efficacy for various types of advanced solid tumors, including non-small cell lung carcinomas (NSCLC). However, its clinical response is often short lived due to the emergence of acquired resistance. The objective of our ongoing project is to identify potential mechanisms underlying the acquisition of sorafenib resistance using in vivo models of A549 human NSCLC xenograft in athymic nude mice. The in vivo sorafenib-resistant tumor model was established by exposing A549 xenografts to once daily treatment with 40 mg/kg of sorafenib for 4 months. Cells isolated from fresh sorafenib-resistant A549 tumors were propagated in vitro and then reimplanted into the same strain of mice. Tumor tissues obtained from the primary and secondary A549 xenografts were subjected to Western blot and immunohistrochemistry analyses. The in vitro study demonstrated that there was no statistically significant difference in the sensitivity to sorafenib treatment between A549 parental cells and tumor cells derived from sorafenib-resistant A549 xenografts. In the in vivo study, relatively accelerated tumor growth rate was observed in 4 out of 10 sorafenib-treated animals bearing the primary A549 xenografts. Re-implantation of tumor cells derived from the sorafenib-resistant A549 xenografts into the secondary mice showed relatively rapid tumor growth in 6 out of 7 sorafenib-treated tumor bearing animals. Preliminary results from the immunofluorescence analysis of frozen tumor sections revealed that the expression of CD31 and α-smooth muscle actin (α-SMA) in sorafenib-resistant primary A549 tumors was significantly reduced compared with the untreated A549 tumors. In contrast, the expression of α-SMA in the secondary sorafenib-resistant A549 tumors was upregulated as compared with that in the primary A549 tumors, while the CD31 expression in the secondary tumors was similar to that in the primary control tumors. Moreover, in the primary A549 tumors, α-SMA immunofluorescence was often observed immediately adjacent to tumor microvessels; whereas in the secondary sorafenib-resistant tumors, α-SMA immunofluorescence mainly appeared in the stromal cells that were involved in the fibrous sheaths surrounding tumor cell nests. Overall, observations from the preliminary study suggest that the secondary sorafenib-resistant tumors have an increased ability to recruit stromal cells, which in turn promote resistance to sorafenib treatment. Further studies are currently underway to explore the potential mechanism associated with the enhanced recruitment of stromal cells in secondary sorafenib-resistant tumors. Citation Format: Qingyu Stephanie Zhou, Xiaofang Guo. Enhanced recruitment of tumor microenvironment components in A549 xenografts with acquired resistance to sorafenib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1387. doi:10.1158/1538-7445.AM2015-1387

Abstract B52: The role of extracellular superoxide dismutase activity in pancreatic cancer biology and therapy

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) cells are known to produce excessive amounts of reactive oxygen species (ROS), particularly superoxide (O2•-), which may contribute to the aggressive nature of this disease. Extracellular superoxide dismutase (EcSOD) is an antioxidant enzyme that catalyzes the dismutation of O2•- to hydrogen peroxide (H2O2) in the extracellular environment. By limiting O2•- concentration and producing H2O2, EcSOD is thought to regulate the redox state of the tumor microenvironment that could contribute to pancreatic cancer progression. The current work tests the hypothesis that EcSOD modulates PDA growth and invasion by modifying the balance between H2O2 and ONOO- formation. Methods: EcSOD and 3-nitrotyrosine (3-NT), a marker of protein nitration by ONOO-, were evaluated in specimens from patients with PDA. EcSOD expression was correlated with clinicopathologic and treatment-related variables and survival in a tissue microarray (TMA) of 114 patients with Stage I-IV PDA. EcSOD was constitutively overexpressed in two primary PDA cell lines and clonogenic survival and doubling time were measured. A Matrigel invasion assay was used to determine the impact of EcSOD on PDA tumor cell invasive capacity. We also utilized and GC4419, a selective SOD mimic currently in clinical development. GC4419 dismutates O2•- but does not react with H2O2 under physiologic conditions. PDA cells stably expressing EcSOD and luciferase were used to create hind leg xenografts in athymic nude mice to assess the impact of EcSOD on tumor growth and then via peritoneal injection to determine the impact on peritoneal metastasis formation using bioluminescence imaging (BLI). Results: EcSOD expression was reduced in PDA compared to normal pancreas in 11/16 specimens evaluated (69%). A decrease in expression was also seen in areas of premalignant ductal epithelium (PanIN-3) relative to adjacent pancreas. In normal pancreas tissue, there was no evidence of immunoreactive 3-NT; however, 11/16 PDA specimens (69%) had robust levels of immunoreactive 3-NT. In the TMA, EcSOD expression was absent in 41/114 of the PDA biopsies (36%). The median survival of patients with intact EcSOD expression was 11.0 months vs. 6.5 months for patients with loss of EcSOD expression. Compared to patients with intact EcSOD, patients with loss of EcSOD more often had advanced stage disease and were less likely to be treated with surgery. In an adjusted proportional hazard model of more than 10 clinical variables, the HR for death with loss of EcSOD was 1.63 (95% CI=1.02-2.58, p=0.04). EcSOD overexpression significantly reduced clonogenic survival, doubling time, and tumor cell invasion relative to controls. GC4419 also significantly reduced PDA invasiveness in the Matrigel assay. PDA cells stably expressing EcSOD showed decreased growth and doubling time in hind limb xenografts and significantly less peritoneal metastasis using BLI. Conclusions: Loss of EcSOD expression and oxidative stress as indicated by the presence of 3-NT is a common finding in PDA. Loss of EcSOD expression correlates with negative prognostic factors and shorter survival in patients with PDA. Overexpression of EcSOD resulted in reduced PDA growth, invasion, and peritoneal metastasis. The selective SOD mimic GC4419 also reduced invasion of PDA tumor cells. Together these findings support the hypothesis that modulating O2•- in the PDA microenvironment alters parameters of progression, oxidative damage, and may represent a target for limiting the spread of PDA. (Supported by The American Surgical Association Foundation Fellowship (JJM) and the American Cancer Society (JJM). Citation Format: James J. Mezhir, Brianne R. O'Leary, Andrew M. Bellizzi, Sean Altekruse, Charles F. Lynch, Brenda Y. Hernandez, Wendy Cozen, Michael D. Henry, Jeffrey Keene, Robert A. Beardsley, Douglas R. Spitz, Frederick E. Domann. The role of extracellular superoxide dismutase activity in pancreatic cancer biology and therapy. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B52.

Bromodomain and extraterminal inhibition blocks tumor progression and promotes differentiation in neuroblastoma

MYCN amplification is a key molecular hallmark of high-risk neuroblastoma. Previously considered an "undruggable" target, MYCN transcription can be disrupted by inhibiting the bromodomain and the extraterminal (BET) domain family of proteins that regulates MYCN transcription epigenetically. JQ1 is a potent, small-molecule BET inhibitor that induces cell-cycle arrest and initiates apoptosis in neuroblastoma. Here, we sought to validate the antitumorigenic effects of JQ1 in neuroblastoma and to evaluate whether blocking N-myc expression with JQ1 promotes neural differentiation. We determined the effects invitro of JQ1 treatment on human neuroblastoma cell growth in both monolayer and sphere-forming conditions. Subcutaneous neuroblastoma xenografts were used for an invivo study. Western blotting and immunohistochemistry were performed to evaluate the effects on neural differentiation and stem cell markers. JQ1 treatment blocked neuroblastoma cell growth in both monolayer and sphere-forming conditions; JQ1 also attenuated the growth of neuroblastoma xenograft in athymic nude mice. Neurofilament expression was enhanced with JQ1 treatment, indicating that JQ1 induces neuronal differentiation. Sphere forming conditions resulted in increased expression of multiple stem cell markers; these effects were reversed with JQ1 treatment. BET inhibition attenuates progression and promotes neural differentiation of neuroblastoma invitro and invivo in mice, providing insight into potential clinical applications of BET inhibitors in the treatment of patients with neuroblastoma.

Honokiol inhibits the growth of head and neck squamous cell carcinoma by targeting epidermal growth factor receptor.

Here, we report the chemotherapeutic effect of honokiol, a phytochemical from Magnolia plant, on human head and neck squamous cell carcinoma (HNSCC). Treatment of HNSCC cell lines from different sub-sites, SCC-1 (oral cavity), SCC-5 (larynx), OSC-19 (tongue) and FaDu (pharynx) with honokiol inhibited their cell viability, which was associated with the: (i) induction of apoptosis, (ii) correction of dysregulatory cell cycle proteins of G0/G1 phase. Honokiol decreased the expression levels of epidermal growth factor receptor (EGFR), mTOR and their downstream signaling molecules. Treatment of FaDu and SCC-1 cell lines with rapamycin, an inhibitor of mTOR pathway, also reduced cell viability of HNSCC cells. Administration of honokiol by oral gavage (100 mg/kg body weight) significantly (P < 0.01-0.001) inhibited the growth of SCC-1 and FaDu xenografts in athymic nude mice, which was associated with: (i) inhibition of tumor cell proliferation, (ii) induction of apoptosis, (iii) reduced expressions of cyclins and Cdks, and (iv) inhibition of EGFR signaling pathway. Molecular docking analysis of honokiol in EGFR binding site indicated that the chemotherapeutic effect of honokiol against HNSCC is mediated through its firm binding with EGFR, which is better than that of gefitinib, a commonly used drug for HNSCC treatment.

Increase in apoptotic effect of Panax ginseng by microwave processing in human prostate cancer cells: in vitro and in vivo studies

BackgroundGinseng, which is widely used in functional foods and as an herbal medicine, has been reported to reduce the proliferation of prostate cancer cells by mechanisms that are not yet fully understood. MethodsThis study was designed to investigate the changes in ginsenoside content in ginseng after treatment with a microwave-irradiation thermal process and to verify the anticancer effects of the extracts. To confirm the anticancer effect of microwave-irradiated processed ginseng (MG), it was tested in three human prostate cancer cell lines (DU145, LNCaP, and PC-3 cells). Involvements of apoptosis and autophagy were assessed using Western blotting. ResultsAfter microwave treatment, the content of ginsenosides Rg1, Re, Rb1, Rc, Rb2, and Rd in the extracts decreased, whereas the content of ginsenosides 20(S)-Rg3, 20(R)-Rg3, Rk1, and Rg5 increased. Antiproliferation results for the human cancer cell lines treated with ginseng extracts indicate that PC-3 cells treated with MG showed the highest activity with an half maximal inhibitory concentration of 48 μg/mL. We also showed that MG suppresses the growth of human prostate cancer cell xenografts in athymic nude mice as an in vivo model. This growth suppression by MG is associated with the inductions of cell death and autophagy. ConclusionTherefore, heat processing by microwave irradiation is a useful method to enhance the anticancer effect of ginseng by increasing the content of ginsenosides Rg3, Rg5, and Rk1.

Discrete Functions Of GSK3α And GSK3β Isoforms In Prostate Tumor Growth And Micrometastasis

ObjectiveRole of glycogen synthase kinase‐3 (GSK3) in cancer is controversial and the isoform specific function of GSK3 in cancer is not well defined. The present study aims to explore the discrete functions of GSK3α and GSK3β in prostate cancer growth and metastasis.&#x0D;Methods and ResultsWe report that silencing the expression of GSK3α or GSK3β isoforms inhibit overall growth and proliferation of the metastatic prostate cancer cells with a predominant role of GSK3α isoform in the process. Deficiency of GSK3α, but not GSK3β inhibited colony formation by the PC3 and DU145 metastatic prostate cancer cells, and the growth of PC3 tumor xenografts in athymic nude mice. Despite the modest effect of GSK3β knockdown on the cell proliferation in vitro, knockdown of GSK3α, but not GSK3β resulted in reduced proliferation and enhanced apoptosis in tumor xenografts in vivo. ShRNA‐mediated knockdown of GSK3α and GSK3β equally inhibited the ability of PC3 cells to migrate and invade the endothelial‐barrier in vitro, and micrometastasis in vivo. Mechanistically, whereas silencing GSK3α resulted in increased expression of pro‐apoptotic markers cleaved caspase‐3 and cleaved caspase‐9 in both PC3 and DU145 cells, silencing GSK3β resulted in the inhibition of cell scattering, establishment of cell‐cell contacts, increased expression and membrane localization of β‐catenin, and reduced expression of epithelial to mesenchymal transition (EMT) markers such as Snail and MMP‐9 indicating specific role for GSK3β in EMT, and acquisition of motility and invasive potential. &#x0D;ConclusionsThe isoform specific role of GSK3α and GSK3β in prostate cancer cells in vitro, and tumor growth and micrometastasis in vivo, via distinct molecular and cellular mechanisms.

Sorafenib and DE605, a novel c-Met inhibitor, synergistically suppress hepatocellular carcinoma.

Sorafenib, an oral multikinase inhibitor of Raf, VEGF and PDGF receptor signaling is approved for advanced hepatocellular carcinoma (HCC). One strategy to improve HCC therapy is to combine agents that target key signaling pathways. Aberrant mesenchymal-epithelial transition factor (c-Met) activation is associated with a variety of human malignancies and therefore represents a target for therapy. In this study, we investigated a novel c-Met inhibitor, DE605, together with sorafenib in hepatocellular carcinoma cells in vitro and in vivo. DE605 and sorafenib synergistically induced apoptosis in hepatocellular carcinoma cells. Mechanistically, DE605 activated the FGFR3/Erk pathway, which in turn was inhibited by sorafenib, resulting in synergism. Finally, DE605 and sorafenib significantly inhibited growth of PLC/PRF/5 hepatocellular carcinoma tumor xenografts in athymic nude mice. Importantly, no obvious weight loss (toxicity) was detected. Thus in combination, DE605 and sorafenib target complementary anti-apoptotic pathways and synergistically suppress HCC, providing the rationale for clinical studies with this novel combination.

Improved anticancer effect of ginseng extract by microwave-assisted processing through the generation of ginsenosides Rg3, Rg5 and Rk1

In this study, we demonstrated the efficient structural conversion of ginsenosides, which enhances the anticancer activity of ginseng by microwave irradiation. The microwave-irradiated product of ginseng (MG) had a higher content of ginsenosides, Rg3+Rg5+Rk1, particularly Rg5 and Rk1, and, thus, has an increased medicinal effect. To confirm the anticancer effect of MG, it was tested in 5 human cancer cell lines (cervical cancer HeLa, gastric cancer AGS, colon cancer HCT-116, lung cancer A549, and liver cancer HepG2 cells). Anti-proliferation results for 5 human cancer cell lines treated with ginseng extracts indicate that HeLa cells treated with MG showed the highest activity with an IC50 value of 130.1 µg/mL. MG also suppressed the growth of human cervical cancer cell xenografts in athymic nude mice as an in vivo model. This growth suppression by MG is associated with the induction of cell death and autophagy. Moreover, there were no toxic sign or decrease in renal and hepatic function in mice administered with MG. Therefore, heat processing by microwave irradiation is a useful method to enhance the anticancer effect of ginseng as it increases the content of ginsenosides, Rg3, Rg5, and Rk1.

Discrete functions of GSK3α and GSK3β isoforms in prostate tumor growth and micrometastasis.

Isoform specific function of glycogen synthase kinase-3 (GSK3) in cancer is not well defined. We report that silencing of GSK3α, but not GSK3β expression inhibited proliferation, survival and colony formation by the PC3, DU145 and LNCaP prostate cancer cells, and the growth of PC3 tumor xenografts in athymic nude mice. Silencing of GSK3α, but not GSK3β resulted in reduced proliferation and enhanced apoptosis in tumor xenografts. ShRNA-mediated knockdown of GSK3α and GSK3β equally inhibited the ability of prostate cancer cells to migrate and invade the endothelial-barrier in vitro, and PC3 cell micrometastasis to lungs in vivo. Mechanistically, whereas silencing GSK3α resulted in increased expression of pro-apoptotic markers cleaved caspase-3 and cleaved caspase-9 in LNCaP, PC3 and DU145 cells, silencing GSK3β resulted in the inhibition of cell scattering, establishment of cell-cell contacts, increased expression and membrane localization of β-catenin, and reduced expression of epithelial to mesenchymal transition (EMT) markers such as Snail and MMP-9. This indicated the specific role of GSK3β in EMT, acquisition of motility and invasive potential. Overall, our data demonstrated the isoform specific role of GSK3α and GSK3β in prostate cancer cells in vitro, and tumor growth and micrometastasis in vivo, via distinct molecular and cellular mechanisms.

A novel PET imaging using ⁶⁴Cu-labeled monoclonal antibody against mesothelin commonly expressed on cancer cells.

Mesothelin (MSLN) is a 40-kDa cell differentiation-associated glycoprotein appearing with carcinogenesis and is highly expressed in many human cancers, including the majority of pancreatic adenocarcinomas, ovarian cancers, and mesotheliomas, while its expression in normal tissue is limited to mesothelial cells lining the pleura, pericardium, and peritoneum. Clone 11-25 is a murine hybridoma secreting monoclonal antibody (mAb) against human MSLN. In this study, we applied the 11-25 mAb to in vivo imaging to detect MSLN-expressing tumors. In in vitro and ex vivo immunochemical studies, we demonstrated specificity of 11-25 mAb to membranous MSLN expressed on several pancreatic cancer cells. We showed the accumulation of Alexa Fluor 750-labeled 11-25 mAb in MSLN-expressing tumor xenografts in athymic nude mice. Then, 11-25 mAb was labeled with 64Cu via a chelating agent DOTA and was used in both in vitro cell binding assay and in vivo positron emission tomography (PET) imaging in the tumor-bearing mice. We confirmed that 64Cu-labeled 11-25 mAb highly accumulated in MSLN-expressing tumors as compared to MSLN-negative ones. The 64Cu-labeled 11-25 mAb is potentially useful as a PET probe capable of being used for wide range of tumors, rather than 18F-FDG that occasionally provides nonspecific accumulation into the inflammatory lesions.

Ursolic Acid Induces Apoptosis of Prostate Cancer Cells via the PI3K/Akt/mTOR Pathway.

Ursolic acid (UA), a pentacyclic triterpenoid, is known to exert antitumor activity in breast, lung, liver and colon cancers. Nonetheless, the underlying mechanism of ursolic acid in prostate cancer cells still remains unclear. In the present study, we report the chemotherapeutic effects of ursolic acid as assessed using in vitro and in vivo models. Treatment of human prostate cancer cells (LNCaP and PC-3) with UA inhibited the proliferation and induced apoptosis in both cell lines as characterized by the increased Annexin V-binding. The induction of apoptosis by UA was associated with a decrease in the levels of Bcl-2, Bcl-xl, survivin, and activated caspase-3. Treatment with UA also inhibited the expression of phosphatidylinositol-3-kinase (PI3K), phosphorylation of Akt and mTOR signaling proteins. Further, administration of UA significantly inhibited the growth of LNCaP prostate tumor xenografts in athymic nude mice, which was associated with inhibition of cell proliferation, induction of apoptosis of tumor cells and decreased expression of PI3K downstream factors, such as p-Akt and p-mTOR in tumor xenograft tissues. Our study demonstrates that UA not only inhibits cell growth but also induces apoptosis through modulation of the PI3K/Akt/mTOR pathway in human prostate cancer cells. We suggest that UA may be a new chemotherapeutic candidate against prostate cancer.

Anthocyanin induces apoptosis of DU-145 cells in vitro and inhibits xenograft growth of prostate cancer.

PurposeTo investigate the effects of anthocyanins extracted from black soybean, which have antioxidant activity, on apoptosis in vitro (in hormone refractory prostate cancer cells) and on tumor growth in vivo (in athymic nude mouse xenograft model).Materials and MethodsThe growth and viability of DU-145 cells treated with anthocyanins were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and apoptosis was assessed by DNA laddering. Immunoblotting was conducted to evaluate differences in the expressions of p53, Bax, Bcl, androgen receptor (AR), and prostate specific antigen (PSA). To study the inhibitory effects of anthocyanins on tumor growth in vivo, DU-145 tumor xenografts were established in athymic nude mice. The anthocyanin group was treated with daily oral anthocyanin (8 mg/kg) for 14 weeks. After 2 weeks of treatment, DU-145 cells (2×106) were inoculated subcutaneously into the right flank to establish tumor xenografts. Tumor dimensions were measured twice a week using calipers and volumes were calculated.ResultsAnthocyanin treatment of DU-145 cells resulted in 1) significant increase in apoptosis in a dose-dependent manner, 2) significant decrease in p53 and Bcl-2 expressions (with increased Bax expression), and 3) significant decrease in PSA and AR expressions. In the xenograft model, anthocyanin treatment significantly inhibit tumor growth.ConclusionThis study suggests that anthocyanins from black soybean inhibit the progression of prostate cancer in vitro and in a xenograft model.

Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer

Human papilloma virus (HPV) is the well-established etiological factor of cervical cancer. E6 and E7 oncoproteins expressed by HPV are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Tanshinone IIA (Tan IIA) is a diterpenoid naphthoquinone found in the traditional Chinese medicine Danshen (Salvia sp.). Tan IIA has been shown to possess anti-tumor activity against several cancer types. In this study we show that Tan IIA potently inhibited proliferation of the human cervical cancer CaSki, SiHa, HeLa and C33a cells. Mechanistically in HPV positive CaSki cells, Tan IIA was found to (i) downregulate expression of HPV E6 and E7 genes and modulate associated proteins E6AP and E2F1, (ii) cause S phase cell cycle arrest, (iii) induce accumulation of p53 and alter expression of p53-dependent targets, (iv) modulate pRb and related proteins, and (v) cause p53-mediated apoptosis by moderating Bcl2, Bax, caspase-3, and PARP cleavage expressions. In vivo, Tan IIA resulted in over 66% reduction in tumor volume of cervical cancer xenograft in athymic nude mice. Tan IIA treated tumor tissues had lower expression of proliferation marker PCNA and changes in apoptosis targets were in agreement with in vitro studies, further confirming reduced proliferation and involvement of multiple targets behind anti-cancer effects. This is the first demonstration of Tan IIA to possess significant anti-viral activity by repressing HPV oncogenes leading to inhibition of cervical cancer. Together, our data suggest that Tan IIA can be exploited as a potent therapeutic agent for the prevention and treatment of cervical and other HPV-related cancers.

Abstract 2999: Interleukin-8 mediates resistance to anti-VEGF therapy in ovarian cancer

Abstract Purpose: Vascular endothelial growth factor (VEGF) families of ligands and receptors are primary signaling pathways in tumor angiogenesis and have been extensively targeted by cancer therapeutics. Ovarian tumors are highly vascularized and high degrees of tumor angiogenesis and VEGF expression in ovarian carcinomas correlate with poorer survival. Encouraging results have been obtained with trials in ovarian cancer that target the VEGF and its receptor. However, the clinical benefits are short-lived, followed by a restoration of tumor progression. Understanding the mechanisms of tumor escape from anti-VEGF therapy is critical to find strategies to overcome the resistance. The purpose of this study was to investigate the potential targets of ovarian cancer resistance to anti-VEGF treatments using experimental phenotypic resistance models. Methods: We generated SKOV3 ovarian cancer xenograft in athymic nude mice and treated with sorafenib (10 mg/kg daily, PO) or bevacizumab (10 mg/kg BIW, IP) over an 8-week period. Treatment-resistance tumors were defined by a long-term trend toward continued tumor progression (&amp;gt; 50% increase from the initial volume) under bevacizumab or sorafenib treatment after an initial response whereas treatment-sensitive tumors were defined by a long-term trend toward stable disease or regression. We performed mouse and human angiogenesis multiplex assay to identify the soluble factor differences in the plasma of resistant and sensitive mice. To examine the role of IL-8 mediated resistance, we disrupted IL-8 function of SKOV3 and HUVEC using pharmacological inhibitors (e.g., anti-IL-8 antibody or IL-8 receptor CXCR2 inhibitor) for in vitro assays of cell growth inhibition, spheroid formation, and cell migration. Further, we evaluated the effectiveness of sorafenib and SB 225002 combination in vivo in SKOV3 xenograft mice. CXCR2 expression was evaluated in human ovarian cancer tissue micro array (TMA). Results: In both sorafenib and bevacizumab resistant mice, IL-8 levels were elevated by 2 to 4 folds compared to their respective sensitive treatment groups in a time-dependent manner. Growth inhibition was significantly higher in SKOV3 (32%, P = 0.002) and HUVEC (25%, P = 0.015) cells when treated with a CXCR2 inhibitor SB 225002 (2 µM) and sorafenib (4 µM) compared to sorafenib alone. SB 225002 (2 µM) disrupted the spheroid formation in SKOV3 cells. SB 225002 and sorafenib combination treatment resulted in 40% less migration of SKOV3 cells when compared to treatment with sorafenib alone. Conclusion: Our results suggest that IL-8 may serve as an important target for sorafenib and bevacizumab resistance in ovarian cancer and disruption of IL-8 activity via CXCR2 inhibition could improve outcomes of anti-VEGF therapy. Citation Format: Bharat Kumar Devapatla, Ankur Sharma, Sukyung Woo. Interleukin-8 mediates resistance to anti-VEGF therapy in ovarian cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2999. doi:10.1158/1538-7445.AM2014-2999