Colorectal Cancer Xenograft Mouse Research Articles

Importance of the fatty acid chain length on in vitro and in vivo anticancer activity of fattigation-platform albumin nanoparticles in human colorectal cancer xenograft mice model

The aims of this study were to design different chain length fatty acid-conjugated albumin nanoparticles (ANPs) and evaluate their anticancer activity in the HCT116 human colorectal cancer xenograft mouse model. Doxorubicin hydrochloride (DOX·HCl) was chosen as a model drug. The different chain lengths of fatty acids (butyric acid; C4, and stearic acid; C18) in albumin conjugates exhibited different physicochemical properties and anticancer activity. Fatty acid-conjugated albumin aided the formation of self-assembled structures with an average size of approximately 200 nm and a negative charge when incubated with excess DOX in an aqueous solution. DOX-loaded long-chain C18-conjugated ANPs allowed efficient encapsulation of hydrophobic DOX into the core of the self-assembled structure, enabling higher drug loading, enhanced colloidal stability and controlled release behavior in PBS pH 7.4 medium as compared with free DOX·HCl or non-fatty acid conjugated ANPs. Furthermore, DOX-loaded fatty acid-conjugated ANPs showed an increased cellular uptake intensity and cytotoxic effects in vitro. In vivo, HCT116 xenograft model experiments confirmed that DOX-loaded C18-conjugated ANPs showed improved anticancer activity and reduced side effects compared with the DOX-treated groups. The long-chain fatty acid-conjugated ANPs synergistically activated the interaction with the free-fatty acid receptor (FFAR) on HCT116 colorectal cancer cells as compared with short-chain C4 or other non-conjugated ANPs. Specifically, DOX-loaded C18-conjugated NPs exhibited significant performance to overexpressed FFAR4 on HCT116 colorectal cancer cells. The fatty acid chain length in the fattigation-platform system could be a promising molecular moiety to improve targeting efficiency and drug accumulation in various cancer therapy.

Indium-111-labeled CD166-targeted peptide as a potential nuclear imaging agent for detecting colorectal cancer stem-like cells in a xenograft mouse model

BackgroundCancer stem cells (CSCs) are involved in drug resistance, metastasis, and relapse of cancers, which can significantly affect tumor therapy. Hence, to develop specifically therapeutic target probe at CSCs for improvement of survival and quality of life of cancer patients is urgently needed. The CD166 protein has been suggested to be involved in colorectal cancer (CRC) tumorigenesis and to be considered a marker for colorectal CSCs (CRCSCs) detection. In this study, therefore, we attend to apply a nuclear imaging agent probe, Glycine18-Cystine-linked CD166-targeted peptides (CD166tp-G18C), to detect the changes of CD166 level in a CRC xenograft mouse model.ResultsWe isolated the CD166-positive cells from the HCT15 CRC cell line (CD166+HCT15) and evaluated their morphology and ability of clone formation, migration, protein expression, and drug resistance. The CD166-positive HCT15 cells display the CSCs characteristics. We discovered and designed a CD166-targeted peptide (CD166tp-G18C) as a targeted probe of CRC stem-like cell for cell binding assay. The CD166tp-G18C confirmed the CD166 protein targeting ability in CD166+HCT15 cells. The diethylenetriaminopentaacetic acid (DTPA)-conjugated CD166tp-G18C further was labeled with indium-111 (111In-DTPA-CD166tp-G18C) as nuclear imaging agent for imaging and bio-distribution analysis in vivo. Finally, we observed that the 111In-DTPA-CD166tp-G18C was significantly enhanced in tumor tissues of CD166+HCT15 xenograft mice as compared to the non-CD166tp-G18C control.ConclusionsOur results indicated that the indium-111-labeled CD166tp-G18C may be served as a powerful tool for colorectal CSCs nuclear imaging in the CRC patients.

Oral Delivery of Multicompartment Nanomedicines for Colorectal Cancer Therapeutics: Combining Loco‐Regional Delivery with Cell‐Target Specificity

AbstractNanomaterials for targeted delivery of chemotherapeutics have received significant attention owing to their potential to enhance the accumulation of therapeutics in diseased tissue. However, in diseases with poor vascularization, such as colorectal cancer (CRC), intravenously injected materials have reduced access to the site of interest. To overcome this challenge, oral administration of targeted nanomedicines is highly desirable. Here, a multicomponent drug delivery system incorporating a degradable alginate microcapsule, formulated to encapsulate micelles targeted to the CD44 receptor is presented. Functional micelles are generated by coupling hyaluronic acid (to target CD44 receptor) to block copolymers of poly(ethylene glycol) monomethyl ether methacrylate and poly(methyl methacrylate). When encapsulated into alginate microcapsules, these micelles form the basis of a novel oral delivery system that offers protection from degradative compartments of the gastrointestinal tract (GIT) and regio‐specific release. The microcapsules demonstrate desirable site‐specific degradation properties in an orthotopic CRC xenograft mouse model, yielding enhanced accumulation of micelles within CD44+ colorectal tumors. The results illustrate that such materials successfully navigate the GIT, regio‐specifically release targeted micelles at the tumor site, and consequently accomplish enhanced accumulation within tumor tissue. Such multi‐component nanomaterials offer a promising means for addressing challenges in treating CRC and difficult to treat diseases.

Abstract A103: Targeting GLI1-mediated NBS1 transcription overcomes 5-FU resistance

Abstract Resistance to radiation and chemotherapy in colorectal patients is considered one of the major contributors of refractory disease and disease progression. Dysregulation or aberrant activation of the SHH/GLI1 signaling pathway, common to both colorectal cancer and other cancer subtypes, influences the integrity of the DNA damage response (DDR) pathway, which may impact tumor response to chemo- and radiation therapies. The DDR pathway is initiated in part by the recognition of a DNA lesion by a sensor protein and the recruitment NBS1 to that lesion. Subsequent repair is initiated by the Mre11, Rad50, NBS1 (MRN) complex formation. In this study, we investigated the expression of GLI1 and NBS1 in tissues samples from 188 patients diagnosed with colorectal cancer by immunohistochemistry and analyzed the clinical significance and prognostic relevance of elevated GLI1-NBS1 co-expression. GLI1 expression was positively associated with the NBS1 expression, and high expression of both in the biopsied specimens significantly correlated with poor patient survival. The result of a series of biochemical experiments directly links GLI1 transcriptional activity to the DNA damage repair pathway. First, NBS1 mRNA levels and protein were found to be reduced upon treatment with GANT61, a GLI inhibitor, indicating that GLI activity was required for NBS1 transcription. Second, overexpression of NBS1 in HT29 cells rescued GANT61-induced cell death by measure of cleaved caspase-3, indicating a significant role of induced DNA damage in the mechanism of GANT61-induced cell death in oncogenic GLI1 cancers. Third, this was verified by ChIP analysis of GLI1 binding to the NBS1 promotor region containing a putative GLI binding sequence. The efficacy of GLI1 inhibition as therapeutic strategy was then tested in parallel with 5-Fluorouracil (5-FU), a component of the standard-of-care therapy FOLFOX. In vitro, colorectal cell lines with GLI1-driven NBS1 expression demonstrated strong 5-FU resistance. These cells were re-sensitized to 5-FU by silencing the expression of NBS1 with siRNA, or with combination therapy involving 5-FU and GANT61. This suggests that a first-in class chemotherapeutic strategy may provide additional treatment options for patients demonstrating 5-FU resistance during the course of their chemotherapy. While GANT61 is commonly used to inhibit GLI in vitro, it exhibits poor pharmacokinetic properties and is not a viable drug for the clinic. To that end, we present a novel GLI1 antagonist SR38832, which exhibits a more stable pharmacokinetic profile than GANT61 in xenograft mouse models and lower IC50 in colon cancer cell lines. In a human colorectal cancer xenograft mouse model, SR38832 significantly inhibited both tumor growth and proliferation. Not only has the identification of SR38832 provided an in vivo tool to study GLI oncogenic activity, but SR38832 is also structural lead to be optimized through an iterative medicinal chemistry approach. Citation Format: Ruowen Zhang, Justin Avery, Jinlu Ma, Theresa Nguyen, Mark J Suto, Bo Xu, Rebecca J Boohaker. Targeting GLI1-mediated NBS1 transcription overcomes 5-FU resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A103. doi:10.1158/1535-7163.TARG-19-A103

Process of immunogenic cell death caused by disulfiram as the anti-colorectal cancer candidate

BackgroundDisulfiram (DSF), a drug widely used to control alcoholism, which has anticancer activity by inducing apoptosis in a copper (Cu)-dependent manner. Numerous evidences from mouse experiments indicated that some anti-cancer agents of chemotherapeutic drugs favor the induction of immunogenic cancer cell death (ICD) leading to tumor-specific immune responses. However, whether DSF could induce the colorectal tumor cells death and the mechanism involved in ICD regulatory remains elusive. The main objective of this study was to elucidate the effect of DSF/Cu on the apoptosis of colorectal cancer (CRC) cells and the expression of the two major ICD markers in CRC cells: calreticulin (CRT) and heat shock proteins (HSP) 70. MethodsFirstly, the toxicity of DSF/Cu in HCT116, SW620 and HCT8 cells was assayed by MTT. Flow cytometry was utilized to detect the apoptosis effects. The effects of DSF/Cu on the expression of ICD-related molecules in tumor tissues were further verified in the CRC xenograft mouse model. ResultsThe results showed that DSF/Cu increase apoptosis of these three cells in a dose dependent manner and significantly inhibited the proliferation at the concentration range from 0.05 to 1.6 μM. Furthermore, the expression of CRT and HSP70 on the cell surface also increased. The rate of transplanted tumors grew slowly, and the expression of CRT and HSP70 in colorectal cancer tissues was increased after treated with DSF/Cu. ConclusionIn conclusion, our results show that DSF/Cu exerts anti-colorectal cancer and its underlying mechanisms are associated with the enhancement of molecules expression of cell ICD. These results provide experimental evidence and theory basis of therapy for developing the DSF/Cu as the drug for CRC.

The exosome secretion inhibitor neticonazole suppresses intestinal dysbacteriosis-induced tumorigenesis of colorectal cancer.

Colorectal cancer (CRC) is the most frequently encountered malignancy associated with the rectum or colon, and accumulating evidences have implicated intestinal dysbacteriosis (IDB, disruption of gut microbiome) and exosomes in the pathology of CRC. We aimed to investigate the effect of IDB on exosome secretion in a CRC xenograft mouse model. An IDB mouse model was established and was inoculated with the CRC cell line SW480 as a xenograft tumor. Tumor growth was monitored for 15days in sham and IDB mice, after which blood was collected to assess serum exosome secretion. A novel exosome secretion inhibitor, neticonazole, was administered to IDB mice bearing CRC xenograft tumors, followed by monitoring of tumor growth and mouse survival. Western blot analysis was performed in xenograft tumors to investigate the underlying molecular mechanism. IDB promoted CRC xenograft tumor growth and exosome secretion, which could be inhibited by the exosome secretion inhibitor neticonazole. Moreover, neticonazole treatment significantly improved the survival of IDB mice with CRC xenograft tumors, likely through increasing apoptosis of CRC xenograft tumor cells. The exosome secretion inhibitor neticonazole may serve as a promising therapeutic candidate against CRC by suppressing IDB-induced CRC tumorigenesis.

Astragaloside IV inhibits human colorectal cancer cell growth.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. However, therapies against CRC have not been completely effective. Astragaloside IV (AS-IV) has shown anti-tumorigenic properties in certain cancers, but its role in CRC remains unclear. In this study, we investigated the therapeutic effect of AS-IV in CRC and explored its underlying mechanism. The results showed that AS-IV dose-dependently inhibited the proliferation of CRC cells and suppressed tumor growth in CRC xenograft mouse models. In addition, AS-IV promoted cell cycle arrest in the G0 phase, associated with increased expression of p21. Furthermore, flow cytometry demonstrated that AS-IV promoted apoptosis of CRC cells in a dose-dependent manner. AS-IV induced caspase-dependent apoptosis, which involved the increased release of cytochrome c and Omi from the mitochondria into the cytoplasm and the up-regulation of Bax/Bcl-2 ratio, as well as the activation of PARP and caspase cascade (caspase-3 and -9). Taken together, our study has identified a novel function of AS-IV and provided a molecular basis for AS-IV's potential applications in the treatment of CRC and other cancers.

MicroRNA-124 inhibits colorectal cancer cell proliferation and suppresses tumor growth by interacting with PLCB1 and regulating Wnt/β-catenin signaling pathway.

Colorectal cancer (CRC) is the most common malignancy for cancer-associated death. This study aimed to investigate the effects of microRNA-124 (miR-124) on tumor proliferation of CRC in vivo and in vitro. MiR-124 mimics were synthesized and transfected into SW620 cells, which were divided into SW620, microRNA-normal control (miR-NC) and miR-124 mimics group. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was used to examine miR-124, chemokine (C-C motif) ligand-20 (CCL20), tankyrase-2 (TNKS2), phospholipase Cbeta1 (PLCB1) and Wnt4. Cell counting kit-8 (CCK-8) was employed to evaluate cell proliferation. The interaction between miR-124 and PLCB1 was tested with the Dual-Luciferase assay. Cell cycle, apoptosis and invasion were also evaluated. CRC xenograft mouse model was established and tumor size was measured. Hematoxylin and eosin (HE) was used to examine inflammation. Western blot was utilized to detect Wnt4. MiR-124 was over-expressed in SW620 cells, significantly reduced CCL20 and enhanced TNKS2 compared to that of the miR-NC group (p<0.05). MiR-124 might play roles by initiating PLCB1 expression. MiR-124 significantly decreased cell viability compared to the miR-NC group (p<0.05). MiR-124 regulated cell cycle and markedly induced apoptosis and inhibited cell invasion compared to the miR-NC group (p<0.05). MiR-124 significantly decreased tumor size of CRC models compared to miR-NC mice (p<0.05). MiR-124 remarkably alleviated inflammation of tumor tissues. MiR-124 markedly enhanced Wnt4 expression compared to the miR-NC group (p<0.05). MiR-124 inhibited tumor cell proliferation in vitro and suppressed tumor growth in vivo by interacting with PLCB1 and regulating the Wnt/β-catenin signaling pathway.

Allosteric Inhibition of Ubiquitin-like Modifications by a Class of Inhibitor of SUMO-Activating Enzyme

Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in thesmall ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation invivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.

Physiologically Based Modeling of the Pharmacokinetics of “Catch-and-Release” Anti-Carcinoembryonic Antigen Monoclonal Antibodies in Colorectal Cancer Xenograft Mouse Models

Engineered monoclonal antibodies (mAbs) with pH-sensitive target release, or “catch-and-release” (CAR) binding, have shown promise in decreasing the extent of target-mediated mAb elimination, increasing mAb exposure, and increasing dose potency. This study developed a mechanistic physiologically based pharmacokinetic (PBPK) model to evaluate the effects of pH-sensitive CAR target binding on the disposition of anti-carcinoembryonic antigen (CEA) mAbs in mouse models of colorectal cancer. The PBPK model was qualified by comparing model-predicted plasma concentration-time data with data observed in tumor-bearing mice following the administration of T84.66, a “standard” anti-CEA mAb that demonstrates strong binding at pH 7.4 and 5.5. Further simulations evaluated the effects CAR pH-dependent binding, with decreasing CEA affinity with decreasing pH, on anti-CEA mAb plasma pharmacokinetics. Simulated data were compared with data observed for a novel CAR mAb, 10H6. The PBPK model provided precise parameter estimates, and excellent data characterization (median prediction error 18.4%) following fitting to T84.66 data. Simulations well predicted 10H6 data (median prediction error 21.4%). Sensitivity analyses demonstrated that key determinants of the disposition of CAR mAbs include the following: antigen binding affinity, the rate constant of mAb-CEA dissociation in acidified endosomes, antigen concentration, and the tumor vasculature reflection coefficient.

PO-198 Flavopereirine suppresses colorectal cancer growth via inducing cell cycle arrest and apoptosis in P53 signalling dependence

Introduction High-grade colorectal cancer (CRC) worldwide is a significant cause of morbidity and mortality. However, the benefit of present therapies are actually limited. Flavopereirine, an alkaloid isolated from Geissospermum vellosii, is shown to suppress the viability of various cancer cells through unknown action. Presently, we investigated the functional mechanism and therapeutic potential of flavopereirine on CRC cells in in vitro and in vivo. Material and methods Effects of flavopereirine on the viability, cell cycle, and apoptosis were evaluated in various CRC cell lines in in vitro, using cell proliferation reagent WST-1, propidium iodide staining, and propidium iodide plus annexin-V double staining, respectively. Protein profiles in cell cycle and apoptosis regulation were verified by Western blotting. Furthermore, CRC cells with p53 null or p53 loss-of-function mutation were applied to investigate the mechanism of flavopereirine-mediated growth inhibition. Moreover, the therapeutic efficacy of flavopereirine on the growth of CRC xenograft mouse model was examined in in vivo. Expressions of P53 and P21 and stimulation of apoptosis by TUNEL staining were further determined in flavopereirine-treated CRC tissues by immunohistochemistry. Results and discussions Our data demonstrated that flavopereirine significantly suppressed viability, caused both intrinsic and extrinsic apoptosis, and induced G2/M-phase cell cycle arrest on CRC cells. On mechanismic study, flavopereirine declined the capacity to suppress the viability and to induce apoptosis in p53 null and p53 loss-of-function mutation CRC cells, indicating the important involvement of P53 activity on flavopereirine-medicated growth suppression. Moreover, flavopereirine significantly repressed the growth of CRC and was followed with the upregulation of P53 and P21 expressions and induction of TUNEL-positive apoptosis in tissues in vivo. Conclusion This study indicates that the therapeutic effect of flavopereirine on suppressing the growth of CRCs is through inhibiting the viability, causing G2/M cell cycle arrest, and inducing apoptosis in the manner of P53 dependence, suggesting that flavopereirine might be a potential drug for treating CRC which carries active P53 expression.

Colorectal cancer cell–derived exosomes containing miR-10b regulate fibroblast cells via the PI3K/Akt pathway

Cancer-associated fibroblasts (CAFs) contribute to the proliferation of colorectal cancer(CRC) cells. However, the mechanism by which CAFs develop in the tumor microenvironment remains unknown. Exosomes may be involved in activating CAFs. Using a miRNA expression profiling array, we determined the miRNA expression profile of secretory exosomes in CRC cells and then identified potential miRNAs with significant differential expression compared to normal cells via enrichment analysis. Predicted targets of candidate miRNAs were then assessed via bioinformatics analysis. Realtime qPCR, western blot, and cell cycle analyses were performed to evaluate the role of candidate exosomal miRNAs. Luciferase reporter assays were applied to confirm whether candidate exosomal miRNAs control target pathway expression. A CRC xenograft mouse model was constructed to evaluate tumor growth in vivo. Exosomes from CRC cells contained significantly higher levels of miR-10b than did exosomes from normal colorectal epithelial cells. Moreover, exosomes containing miR-10b were transferred to fibroblasts. Bioinformatics analysis identified PIK3CA, as a potential target of miR-10b. Luciferase reporter assays confirmed that miR-10b directly inhibited PIK3CA expression. Co-culturing fibroblasts with exosomes containing miR-10b significantly suppressed PIK3CA expression and decreased PI3K/Akt/mTOR pathway activity. Finally, exosomes containing miR-10b reduced fibroblast proliferation but promoted expression of TGF-β and SM α-actin, suggesting that exosomal miR-10b may activate fibroblasts to become CAFs that express myofibroblast markers. These activated fibroblasts were able to promote CRC growth in vitro and in vivo. CRC-derived exosomes actively promote disease progression by modulating surrounding stromal cells, which subsequently acquire features of CAFs.

Self-assembling toxin-based nanoparticles as self-delivered antitumoral drugs

Loading capacity and drug leakage from vehicles during circulation in blood is a major concern when developing nanoparticle-based cell-targeted cytotoxics. To circumvent this potential issue it would be convenient the engineering of drugs as self-delivered nanoscale entities, devoid of any heterologous carriers. In this context, we have here engineered potent protein toxins, namely segments of the diphtheria toxin and the Pseudomonas aeruginosa exotoxin as self-assembling, self-delivered therapeutic materials targeted to CXCR4+ cancer stem cells. The systemic administration of both nanostructured drugs in a colorectal cancer xenograft mouse model promotes efficient and specific local destruction of target tumor tissues and a significant reduction of the tumor volume. This observation strongly supports the concept of intrinsically functional protein nanoparticles, which having a dual role as drug and carrier, are designed to be administered without the assistance of heterologous vehicles.

Cancer-Type OATP1B3 mRNA in Extracellular Vesicles as a Promising Candidate for a Serum-Based Colorectal Cancer Biomarker.

Cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA is a variant isoform of the liver-type OATP1B3. Because Ct-OATP1B3 mRNA shows an excellent cancer-specific expression profile in colorectal cancer (CRC), and that its expression levels are associated with CRC prognosis, it holds the potential to become a useful CRC detection and diagnosis biomarker. While the potential is currently justified only at the tissue level, if existence of Ct-OATP1B3 mRNA in CRC-derived extracellular vesicles (EVs) is validated, the findings could enhance its translational potential as a CRC detection and diagnosis biomarker. Therefore, this study aims at proving that Ct-OATP1B3 mRNA exists in CRC-derived EVs, and can be detected using serum specimens. To examine the possibility of Ct-OATP1B3 mRNA being existed in extracellular milieu, we isolated EVs from the human CRC (HCT116, HT-29, and SW480) cell lines, and prepared their cDNAs. The RT-PCR results showed that Ct-OATP1B3 mRNA was clearly present in EVs derived from the human CRC cell lines. Then, in order to further explore the possibility that Ct-OATP1B3 mRNA in CRC-derived EVs can be detected in serum, we isolated serum EVs derived from human CRC xenograft mice, and then performed RT-PCR. The results showed that Ct-OATP1B3 mRNA could be found in all serum EV and CRC tissue samples of the mice examined. Collectively, our findings, which show that Ct-OATP1B3 mRNA exists in EVs and can be detected in (at least) mouse serum, strengthen the potential use of Ct-OATP1B3 mRNA as a serum-based CRC biomarker.

Therapeutic inhibition of SGK1 suppresses colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death worldwide. Thus, the development of new therapeutic targets for CRC treatment is urgently needed. SGK1 is involved in various cellular activities, and its dysregulation can result in multiple cancers. However, little is known about its roles and associated molecular mechanisms in CRC. In present study, we found that SGK1 was highly expressed in tumor tissues compared with peri-tumor samples from CRC patients. In vitro experiments revealed that SGK1 overexpression promoted colonic tumor cell proliferation and migration and inhibited cell apoptosis induced by 5-fluorouracil (5-FU), while SGK1 shRNA and inhibitors showed the inverse effects. Using CRC xenograft mice models, we demonstrated that knockdown or therapeutic inhibition of SGK1 repressed tumor cell proliferation and tumor growth. Moreover, SGK1 inhibitors increased p27 expression and promoted p27 nuclear accumulation in colorectal cancer cells, and p27 siRNAs could attenuate the repression of CRC cell proliferation induced by SGK1 inhibitors. Collectively, SGK1 promotes colorectal cancer development via regulation of CRC cell proliferation, migration and survival. Inhibition of SGK1 represents a novel strategy for the treatment of CRC.

Scutellaria barbata D. Don inhibits colorectal cancer growth via suppression of Wnt/β-catenin signaling pathway

To investigate the effect of the ethanol extract of Scutellaria barbata D. Don (EESB) on colorectal cancer (CRC) growth and Wnt/β-catenin signaling pathway in vivo and in vitro. In vivo experiment, CRC xenograft mouse model was constructed with injection of HT-29 cells. Following xenograft implantation, twenty mice were randomly divided into EESB-treated group (n=10) and control group (n=10) by a random number table, and were given with intra-gastric administration of 2 g/kg EESB or saline, 5 days a week for 16 days, respectively. At the end of experiment, tumors were removed and weighed by electronic scales. The proliferation biomarker Ki-67 of tumor was evaluated by immunohistochemistry (IHC) assay. In vitro study, HT-29 cells were treated with 0, 0.5, 1.5, 2.5 mg/mL EESB for 24 h. At the end of the treatment, the viability and survival of HT-29 cells were determined by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and colony formation assay, respectively. The mRNA expression of c-Myc, Survivin and adenomatous polyposis coli (APC) was examined by reverse transcription-polymerase chain reaction (RT-PCR) both in tumor tissues of CRC xenograft mice and HT-29 cells. Protein expression of c-Myc, Survivin, APC, and β-catenin as well as β-catenin phosphorylation level were evaluated by IHC assay or Western blotting. EESB significantly reduced tumor weight in CRC xenografts mice, compared with the control group (P<0.05). IHC assay showed that EESB significantly inhibited protein expression of Ki-67 in tumor tissues (P<0.05). MTT assay showed that EESB significantly reduced HT-29 cell viability in a dose-dependent manner (P<0.05). Colony formation assay showed that EESB dose-dependently decreased the survival of HT-29 cells (P<0.05). In addition, RT-PCR assay showed that EESB decreased the mRNA expression of c-Myc and Survivin and increased APC expression, both in tumor tissues of CRC xenograft mice and HT-29 cells (P<0.05). IHC assay or Western blotting showed that EESB decreased protein expression of β-catenin, c-Myc and Survivin, as well as increased APC expression and β-catenin phosphorylation in tumor tissues or HT-29 cells (P<0.05). EESB significantly reduced tumor growth in CRC xenografts mice, and inhibited the viability and survival of HT-29 cells. EESB could suppress the activation of the Wnt/β-catenin pathway, which might be one of the mechanisms whereby Scutellaria barbata D. Don exerts its anticancer activity.

Estrogen Activation by Steroid Sulfatase Increases Colorectal Cancer Proliferation via GPER.

Context:Estrogens affect the incidence and progression of colorectal cancer (CRC), although the precise molecular mechanisms remain ill-defined.Objective:The present study investigated prereceptor estrogen metabolism through steroid sulphatase (STS) and 17β-hydroxysteroid dehydrogenase activity and subsequent nongenomic estrogen signaling in human CRC tissue, in The Cancer Genome Atlas colon adenocarcinoma data set, and in in vitro and in vivo CRC models. We aimed to define and therapeutically target pathways through which estrogens alter CRC proliferation and progression.Design, Setting, Patients, and Interventions:Human CRC samples with normal tissue-matched controls were collected from postmenopausal female and age-matched male patients. Estrogen metabolism enzymes and nongenomic downstream signaling pathways were determined. CRC cell lines were transfected with STS and cultured for in vitro and in vivo analysis. Estrogen metabolism was determined using an ultra-performance liquid chromatography–tandem mass spectrometry method.Primary Outcome Measure:The proliferative effects of estrogen metabolism were evaluated using 5-bromo-2′-deoxyuridine assays and CRC mouse xenograft studies.Results:Human CRC exhibits dysregulated estrogen metabolism, favoring estradiol synthesis. The activity of STS, the fundamental enzyme that activates conjugated estrogens, is significantly (P < 0.001) elevated in human CRC compared with matched controls. STS overexpression accelerates CRC proliferation in in vitro and in vivo models, with STS inhibition an effective treatment. We defined a G-protein–coupled estrogen receptor (GPER) proproliferative pathway potentially through increased expression of connective tissue growth factor in CRC.Conclusion:Human CRC favors estradiol synthesis to augment proliferation via GPER stimulation. Further research is required regarding whether estrogen replacement therapy should be used with caution in patients at high risk of developing CRC.

Curative Multicycle Radioimmunotherapy Monitored by Quantitative SPECT/CT-Based Theranostics, Using Bispecific Antibody Pretargeting Strategy in Colorectal Cancer

Radioimmunotherapy of solid tumors using antibody-targeted radionuclides has been limited by low therapeutic indices (TIs). We recently reported a novel 3-step pretargeted radioimmunotherapy (PRIT) strategy based on a glycoprotein A33 (GPA33)-targeting bispecific antibody and a small-molecule radioactive hapten, a complex of 177Lu and S-2-(4-aminobenzyl)-1,4,7,10-tetraazacyclododecane tetraacetic acid (177Lu-DOTA-Bn), that leads to high TIs for radiosensitive tissues such as blood (TI = 73) and kidney (TI = 12). We tested our hypothesis that a fractionated anti-GPA33 DOTA-PRIT regimen calibrated to deliver a radiation absorbed dose to tumor of more than 100 Gy would lead to a high probability of tumor cure while being well tolerated by nude mice bearing subcutaneous GPA33-positive SW1222 xenografts. Methods: We treated groups of nude mice bearing 7-d-old SW1222 xenografts with a fractionated 3-cycle anti-GPA33 DOTA-PRIT regimen (total administered 177Lu-DOTA-Bn activity, 167 MBq/mouse; estimated radiation absorbed dose to tumor, 110 Gy). In randomly selected mice undergoing treatment, serial SPECT/CT imaging was used to monitor treatment response and calculate radiation absorbed doses to tumor. Necropsy was done on surviving animals 100-200 d after treatment to determine frequency of cure and assess select normal tissues for treatment-related histopathologies. Results: Rapid exponential tumor progression was observed in control treatment groups (i.e., no treatment or 177Lu-DOTA-Bn only), leading to euthanasia due to excessive tumor burden, whereas 10 of 10 complete responses were observed for the DOTA-PRIT-treated animals within 30 d. Treatment was well tolerated, and 100% histologic cure was achieved in 9 of 9 assessable animals without detectable radiation damage to critical organs, including bone marrow and kidney. Radiation absorbed doses to tumor derived from SPECT/CT (102 Gy) and from biodistribution (110 Gy) agreed to within 6.9%. Of the total dose of approximately 100 Gy, the first dose contributes 30%, the second dose 60%, and the third dose 10%. Conclusion: In a GPA33-positive human colorectal cancer xenograft mouse model, we validated a SPECT/CT-based theranostic PRIT regimen that led to 100% complete responses and 100% cures without any treatment-related toxicities, based on high TIs for radiosensitive tissues. These studies support the view that anti-GPA33 DOTA-PRIT will be a potent radioimmunotherapy regimen for GPA33-positive colorectal cancer tumors in humans.

The VEGFR Inhibitor Cediranib Improves the Efficacy of Fractionated Radiotherapy in a Colorectal Cancer Xenograft Model

Background/Purpose: Radiotherapy (RT) increases local tumor control in locally advanced rectal cancer, but complete histological response is seen in only a minority of cases. Antiangiogenic therapy has been proposed to improve RT efficacy by “normalizing” the tumor microvasculature. Here, we examined whether cediranib, a pan-vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor, improves microvascular function and tumor control in combination with RT in a mouse colorectal cancer (CRC) model. Methods: CRC xenografts (HT29) were grown subcutaneously in mice. Animals were treated for 5 consecutive days with vehicle, RT (1.8 Gy daily), cediranib (6 mg/kg po), or combined therapy (cediranib 2 h prior to radiation). Tumor volume was measured with calipers. Vascular changes were analyzed by dynamic contrast-enhanced MRI, oxygenation and interstitial fluid pressure probes and histology. To investigate vascular changes more in detail, a second set of mice were fitted with titanium dorsal skinfold window chambers, wherein a HT29 tumor cell suspension was injected. In vivo fluorescence microscopy was performed before and after treatment (same treatment protocol). Results: In vivo microscopy analyses showed that VEGFR inhibition with cediranib led to a “normalization” of the vessel wall, with decreased microvessel permeability (p < 0.0001) and tortuosity (p < 0.01), and a trend to decreased vessel diameters. This seemed to lead to lower tumor hypoxia rates in the cediranib and combination groups compared to the control and RT groups. This led to an increased tumor control in the combination group compared to controls or monotherapy (p < 0.0001). Conclusions: The combination of RT with cediranib enhances tumor control in a CRC xenograft mouse model. Microvascular analyses suggest that cediranib leads to vascular normalization and improved oxygenation.

Oleanolic acid inhibits colorectal cancer angiogenesis in vivo and in vitro via suppression of STAT3 and Hedgehog pathways.

Angiogenesis is an essential process of cancer progression and is regulated by multiple intracellular signaling pathways, including signal transducer and activator of transcription3 (STAT3) and sonic hedgehog (SHH). Thus, these pathways have become a promising target for anti‑cancer therapeutic strategies. Oleanolic acid (OA) is an active compound present in various herbal medicines, which have been used historically for the clinical treatment of various types of human malignancies, including colorectal cancer (CRC). The present study used a CRC mouse xenograft model and human umbilical vein endothelial cells (HUVECs) to evaluate the effect of OA on tumor angiogenesis and on the activation of the STAT3 and SHH signaling pathways. It was determined that OA treatment significantly inhibited tumor growth and reduced intratumoral microvessel density (MVD) in CRC mice. In addition, OA treatment inhibited the proliferation, migration and tube formation in HUVECs, in a dose and time-dependent manner. Furthermore, OA markedly suppressed the activation of the STAT3 and SHH signaling pathways and inhibited the expression of the pro‑angiogenic vascular endothelial growth factorA and basic fibroblast growth factor, two important target genes of the aforementioned signaling pathways. Therefore it is suggested that inhibition of tumor angiogenesis via the suppression of multiple signaling pathways may be one of the underlying mechanisms by which OA exerts its anti-cancer effect.