Human Colon Cancer Xenografts Research Articles

In Vitro and in Vivo antitumor activity and the mechanism of siphonodictyal B in human colon cancer cells.

Liphagal, isolated from the marine sponge Aka coralliphaga, exhibits phosphatidylinositol 3‐kinase alpha (PI3Kα) inhibitory activity and cytotoxic effects in human cancer cells. Siphonodictyal B, the biogenetic precursor of liphagal, also has PI3K inhibitory activity. However, its cytotoxic or antitumor activities have not been evaluated. In this study, we demonstrated that siphonodictyal B inhibits several kinases such as CDK4/6, CDK7, and PIM2 in addition to PI3K in vitro and that siphonodictyal B exhibits more potent cytotoxic effects than liphagal against human colon cancer cell lines. Furthermore, treatment with siphonodictyal B resulted in increased PARP cleavage, a larger sub‐G1 fraction, and a larger annexin V‐positive cell population, all of which are indicative of apoptosis induction. As a mechanism of apoptosis induction, we found that siphonodictyal B activates the p38 MAPK pathway, leading the upregulation of proapoptotic factors. Moreover, siphonodictyal B increased ROS levels, thus promoting p38 MAPK pathway activation. NAC, an ROS scavenger, almost completely reversed both the cytotoxic and p38 MAPK pathway‐activating effects of siphonodictyal B. These results indicate that the p38 MAPK pathway might be involved downstream of ROS signaling as part of the mechanism of siphonodictyal B‐induced apoptosis. Finally, siphonodictyal B displayed antitumor effects in a human colon cancer xenograft mouse model and increased p38 phosphorylation in tumor tissue. These results suggest that siphonodictyal B could serve as the basis of a novel anticancer drug.

Evaluation of Anti-Tumorigenic Effects of Diosmetin against Human Colon Cancer Xenografts in Athymic Nude Mice.

Colon cancer is the third most common type of cancer in the world. Diosmetin (Dis), a natural O-methylated flavone, has been reported to have anti-cancer effects against different types of cancer. Although the mechanisms of action of Dis against several cancer cell lines are well reported, in vivo anti-tumorigenesis properties of this compound are still obscure. Therefore, this study aimed to investigate the anti-tumorigenesis properties of Dis against HCT-116 colon cancer xenografts in nude mice. HCT-116 colon cancer cells were injected in NCr nu/nu nude mice and treatment with Dis was initiated after the tumor volumes reached 100 mm3 and continued for four weeks. On the sacrificing date nude mice treated with 100 mg/kg of Dis showed significant lower tumor volume (264 ± 238.3 mm3) as compared to the untreated group (1428.8 ± 459.6 mm3). Anti-apoptotic Bcl-2 protein was significantly downregulated, while apoptotic protein (Bax) was significantly overexpressed in nude mice treated with 100 mg/kg Dis as compared to untreated mice. In conclusion, our in vivo results indicate that Dis significantly reduces tumor growth rate of HCT-116 colon cancer cells in nude mice at a dose of 100 mg/kg, and has no toxic effects in ICR mice up to 2000 mg/kg.

Abstract 3498: Highly specific macrocyclic ATR inhibitors for the targeted treatment of a broad spectrum of cancers showing lack of anemia or neutropenia in pre-clinical animal models

Abstract Ataxia Telangiectasia and Rad3-related (ATR) and its downstream effector Checkpoint Kinase 1 (CHK1) are central to the protection of stalled replication forks.Specific targeting of the ATR is synthetically lethal with multiple cancer-associated changes including oncogenic stress and defects in the DDR pathway and represents an emerging strategy to treat a broad spectrum of cancers. Atrin Pharmaceuticals has rationally designed a novel series of conformationally constrained macrocyclic ATR inhibitors with higher potency and selectivity than other ATR inhibitors currently in clinical development. An in-depth characterization of the ATRN series identified ATRN-119 as our lead compound with an in vitro enzyme IC50 of 20 nM and inhibition of ATR substrate CHK1 Ser345 phosphorylation in cells at an IC50 of 5 nM. ATRN-119 inhibits ATR in cells at much lower concentrations and demonstrates higher selectivity (>2000) for ATR over other closely-related PIK-kinases like ATM, DNA-PK and mTOR. Additionally, ATRN series have favorable ADME properties with increased water solubility and metabolic stability in human serum of up to 4 hours. Oral dosing of ATRN-119 showed significant antitumor effects in human pancreatic and colon cancer xenografts and in orthotopic ovarian Patient Derived Xenograft (PDX) tumors. Notably, hematological analysis of mice treated with daily oral dosing of ATRN-119 indicated no thrombocytopenia, anemia or neutropenia up to 4 weeks of treatment. Exploratory multiple high dosing toxicity studies in rats and dogs indicate significant exposure and good tolerability with lack of anemia or neutropenia. In a series of in-vitro cell viability assays, three dimensional organoid cultures and in-vivo combinationstudies, ATRN-119 showed significant synergism with various PARP inhibitors as well as restoration of PARPi sensitivity in PARPi resistant tumors. Our data suggests a new generation of highly potent and selective ATR inhibitors with favorable safety profile and a broad clinical therapeutic potential either as monotherapy, in combination with PARP inhibitors or as a synthetic lethal approach with key DDR mutations. Citation Format: Sahithi Pamarthy, Dansu Li, Ekaterine Goliadze, Tina Gill, Lanqi Jia, Erin George, Laura R. Butler, Ryan L. Ragland, Michel Afargan, Fiona A. Simpkins, Eric J. Brown, Oren Gilad. Highly specific macrocyclic ATR inhibitors for the targeted treatment of a broad spectrum of cancers showing lack of anemia or neutropenia in pre-clinical animal models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3498.

1-Phenyl-dihydrobenzoindazoles as novel colchicine site inhibitors: Structural basis and antitumor efficacy

The colchicine site inhibitors (CSIs) showed promising prospects as antitumor agents due to their vascular disrupting activities besides antimitotic activities. 1-Phenyl-dihydrobenzoindazole was found as a novel scaffold of CSI without the cis−trans isomerization problem. The X-ray co-crystal structure of the lead compound with tubulin was determined, which revealed the binding mode including special water-bridged hydrogen bonds. The structure also provided guidance for the structural optimization of this type of CSI, which led to the discovery of the most potent inhibitor A3, with growth IC50 lower than 1 nM against human colon cancer cell lines and tubulin polymerization IC50 of 1.6 μM. In addition, its water-soluble prodrug B1 showed good in vivo antitumor activity on two human colon cancer xenograft nude mice models, encouraging further study of this type of antitumor compound.

A novel mouse model of sporadic colon cancer induced by combination of conditional Apc genes and chemical carcinogen in the absence of Cre recombinase.

Although valuable insights into colon cancer biology have been garnered from human colon cancer cell lines and primary colonic tissues, and animal studies using human colon cancer xenografts, immunocompetent mouse models of spontaneous or chemically induced colon cancer better phenocopy human disease. As most sporadic human colon tumors present adenomatous polyposis coli (APC) gene mutations, considerable effort has gone into developing mice that express mutant Apc alleles that mimic human colon cancer pathogenesis. A serious limitation of many of these Apc-mutant murine models, however, is that these mice develop numerous tumors in the small intestine but few, if any, in the colon. In this work, we examined three spontaneous mouse models of colon tumorigenesis based upon the widely used multiple intestinal neoplasia (Min) mouse: mice with either constitutive or conditional Apc mutations alone or in combination with caudal-related homeobox transcription factor CDX2P-Cre transgene - either with or without exposure to the potent colon carcinogen azoxymethane. Using the CDX2 promoter to drive Cre recombinase transgene expression effectively inactivated Apc in colonocytes, creating a model with earlier tumor onset and increased tumor incidence/burden, but without the Min mouse model's small intestine tumorigenesis and susceptibility to intestinal perforation/ulceration/hemorrhage. Most significantly, azoxymethane-treated mice with conditional Apc expression, but absent the Cre recombinase gene, demonstrated nearly 50% tumor incidence with two or more large colon tumors per mouse of human-like histology, but no small intestine tumors - unlike the azoxymethane-resistant C57BL/6J-background Min mouse model. As such this model provides a robust platform for chemoprevention studies.

Oncolytic properties of non-vaccinia poxviruses.

Vaccinia virus, a member of the Poxviridae family, has been extensively used as an oncolytic agent and has entered late stage clinical development. In this study, we evaluated the potential oncolytic properties of other members of the Poxviridae family. Numerous tumor cell lines were infected with ten non-vaccinia poxviruses to identify which virus displayed the most potential as an oncolytic agent. Cell viability indicated that tumor cell lines were differentially susceptible to each virus. Raccoonpox virus was the most potent of the tested poxviruses and was highly effective in controlling cell growth in all tumor cell lines. To investigate further the oncolytic capacity of the Raccoonpox virus, we have generated a thymidine kinase (TK)-deleted recombinant Raccoonpox virus expressing the suicide gene FCU1. This TK-deleted Raccoonpox virus was notably attenuated in normal primary cells but replicated efficiently in numerous tumor cell lines. In human colon cancer xenograft model, a single intratumoral inoculation of the recombinant Raccoonpox virus, in combination with 5-fluorocytosine administration, produced relevant tumor growth control. The results demonstrated significant antitumoral activity of this new modified Raccoonpox virus armed with FCU1 and this virus could be considered to be included into the growing armamentarium of oncolytic virotherapy for cancer.

Pharmacokinetic Modeling of Targeted Ultrasound Contrast Agents for Quantitative Assessment of Anti-Angiogenic Therapy: a Longitudinal Case-Control Study in Colon Cancer

PurposeTo evaluate quantitative and semi-quantitative ultrasound molecular imaging (USMI) for antiangiogenic therapy monitoring in human colon cancer xenografts in mice.ProceduresColon cancer was established in 17 mice by injection of LS174T (Nr = 9) or CT26 (Nn = 8) cancer cells to simulate clinical responders and non-responders, respectively. Antiangiogenic treatment (bevacizumab; Nrt = Nnt = 5) or control treatment (saline; Nrc = 4, Nnc = 3) was administered at days 0, 3, and 7. Three-dimensional USMI was performed by injection at days 0, 1, 3, 7, and 10 of microbubbles targeted to the vascular endothelial growth factor receptor 2 (VEGFR2). Microbubble binding rate (kb), estimated by first-pass binding model fitting, and semi-quantitative parameters late enhancement (LE) and differential targeted enhancement (dTE) were compared at each day to evaluate their ability to assess and predict the response to therapy. Correlation analysis with the ex-vivo immunohistological quantification of VEGFR2 expression and the percentage blood vessel area was also performed.ResultsSignificant changes in the USMI parameters during treatment were observed only in the responders treated with bevacizumab (p-value < 0.05). Prediction of the response to therapy as early as 1 day after treatment was achieved by the quantitative parameter kb (p-value < 0.01), earlier than possible by tumor volume quantification. USMI parameters could significantly distinguish between clinical responders and non-responders (p-value << 0.01) and correlated well with the ex-vivo quantification of VEGFR2 expression and the percentage blood vessels area (p-value << 0.01).ConclusionUSMI (semi)quantitative parameters provide earlier assessment of the response to therapy compared to tumor volume, permit early prediction of non-responders, and correlate well with ex-vivo angiogenesis biomarkers.

Antitumor activity of RT2 peptide derived from crocodile leukocyte peptide on human colon cancer xenografts in nude mice.

RT2, derived from the leukocyte peptide of Crocodylus siamensis, can kill human cervical cancer cells via apoptosis induction, but no evidence has shown in vivo. In this study, we investigated the antitumor effect of RT2 on human colon cancer xenografts in nude mice. Twenty-four mice were injected subcutaneously with human colon cancer HCT 116 cells. Eleven days after cancer cell implantation, the mice were treated with intratumoral injections of phosphate buffered saline (PBS) or RT2 (0.01, 0.1, and 1 mg/mouse) once every 2 days for a total of 5 times. The effect of a 10-day intratumoral injection of RT2 on body weight, biochemical, and hematological parameters in BALB/c mice showed no significant difference between the groups. Tumor volume showed a significant decrease only in the treatment group with RT2 (1 mg/mouse) at day 6 (P < .05), day 8 (P < .01), and day 10 (P < .01) after the first treatment. The protein expression levels of cleaved poly (ADP-ribose) polymerase (PARP), apoptosis-inducing factor (AIF), and the p53 tumor suppressor protein (p53) in xenograft tumors increased after treatment with RT2 (1 mg/mouse) compared to those in the PBS-injected group. Moreover, RT2 increased the expression of Endo G and Bcl-2 family proteins. Therefore, the peptide RT2 can inhibit tumor growth via the induction of apoptosis in an in vivo xenograft model.

PO-058 Unravelling the protective role of androgens/androgen receptorin breast cancer: when BAD goes good

IntroductionThe transcription factor STAT6 is strongly expressed in various tumours with highest expression in malignant lymphomas and pancreatic, colorectal, prostate and breast cancers. STAT6 expression in colorectal cancer is associated with an increased malignancy and a poor prognosis. Incidence of colorectal cancer is approximately 1,361,000 patients per annum worldwide and approximately 60% show STAT6 expression. Patients with colorectal cancer expressing STAT6 also show poor survival rates. The 5 year relative survival rate for patients with stage IIIC and IV colon cancer is about 53% and 11% respectively. Techniques aimed at reducing or blocking STAT6 expression may be useful in treating those cancers with STAT6 expression.Material and methodsCelixir owns a patent covering four STAT6 specific small interfering RNA (siRNA) sequences. We tested these four sequences in vitro using the human colon adenocarcinoma cell line HT-29. The four sequences were introduced individually and in combination into HT-29 cells by cationic lipid-mediated transfer at different concentrations (10 to 200 nM). STAT6 mRNA and protein levels were analysed to confirm the transfection was successful. The proliferation of cells was analysed using cell counting at various times (using a NucleoCounter) and the apoptosis of cells was analysed using Annexin V and propidium iodide (PI) staining.Results and discussions100 nM siRNA concentration was the most effective and all four individual sequences knocked-down STAT6 mRNA and protein by more than 50%. All individual sequences were capable of significantly inhibiting cell proliferation, with sequences #1 and #4 reducing the number of cells to ~50% compared with control 7 days after treatment. STAT6 silencing also significantly induced late apoptotic events. Despite using several combinations of all four sequences at the same time and serially, the best results were obtained with sequences #1 and #4 individually.ConclusionThesein vitro resultsdemonstrate that the STAT6 siRNA sequences are capable of inhibiting theproliferation of and inducing late apoptosis of HT-29 colon cancer cells and,in some instances, halving the number of cancer cells. We propose to repeatthese experiments in vivousing immunocompromised mice containing human colon cancer xenografts and totest the siRNA sequences in other cancers in which STAT6 is expressed.

Abstract 5920: Antitumor efficacy of a dual stromal and tumor targeted oncolytic measles virus in breast and colon cancer models

Abstract Efficient systemic delivery of oncolytic viruses remains an unresolved issue and an obstacle to the success of oncolytic virotherapy. One of the main barriers that lead to decreased oncolytic viral delivery to tumor cells is the tumor stromal component. A potential strategy to circumvent this problem is to use a retargeted oncolytic virus that is able to target, and replicate in tumor stromal cells. The aim of this study is to evaluate the consequences of tumor stromal/vascular targeting by oncolytic measles virus via urokinase receptor (uPAR) as they relate to improved viral delivery and antitumor efficacy. We rescued and characterized a novel dual targeting MV-un-muPA, which binds human cancer cells via CD46 and murine tissues via murine uPAR. This novel virus variant was generated as a tool to investigate the ability of a virus to target tumor stromal cells, especially tumor microvasculature (via murine uPAR) and deliver the infection to tumor cells (via CD46) in vivo. In vitro, MV-un-muPA infected and induced cytotoxicity to human or murine cancer cells in a species specific manner. Efficient MV-un-muPA replication was demonstrated in multiple human and murine cancer cells as well as murine fibroblasts in vitro. MV-un-muPA mediated viral transfer was demonstrated between murine endothelial or murine fibroblast to (RFP expressing) human cancer cells. To further validate the consequences of tumor stromal/vascular targeting by oncolytic measles virus via uPAR as they relate to improved viral delivery, the effects of MV-un-muPA vs a non-targeted MV virus (MV-GFP) were assessed in human breast (MDA-MD231) and colon (HT-29) cancer xenograft models. In these models, tumor cells express human CD46 and the host stroma expresses murine uPAR. Tumor bearing mice were treated with MV-GFP (targeting tumor cells via CD46), dual targeting virus MV-un-muPA (target tumor stroma via murine uPAR, and tumor cells via CD46) by intravenous administration (3 doses). Systemic administration of MV-un-muPA resulted in a significant delay in tumor progression as well as improved survival compared to mice treated with vehicle control or MV-GFP. Tumor based studies showed increased levels of viral RNA, tumor apoptosis, resulted in a significantly reduced stroma and the reduction of cancer associated fibroblasts and endothelial cells, in tumors treated with the dual targeted virus, compared to MV-GFP. In conclusion, our results demonstrate for the first time that dual targeting of tumor stroma and tumor cells by MV-un-m-uPA enhance in vivo viral delivery and oncolytic virus antitumor efficacy in two in vivo models of cancer. This serves as clear proof that vascular/stromal targeting is an effective strategy to enhance viral delivery and antitumor effects and support future studies to further uncover the potential of stromal targeted oncolytic viruses. Citation Format: Yuqi Jing, Valery Chavez, Natasha Karishma Khatwani, Jaime Merchan. Antitumor efficacy of a dual stromal and tumor targeted oncolytic measles virus in breast and colon cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5920.

Development and Characterization of a Novel Congenic Rat Strain for Obesity and Cancer Research

ABSTRACTThe association between a Western Diet and colon cancer suggests that dietary factors and/or obesity may contribute to cancer progression. Our objective was to develop a new animal model of obesity and the associated pathophysiology to investigate human cancer independent of dietary components that induce obesity.A novel congenic rat strain was established by introducing the fa allele from the Zucker rat into the Rowett Nude rat to generate a “fatty nude rat”. The obese phenotype was first characterized in the new model. To then examine the utility of this model, lean and obese rats were implanted with HT-29 human colon cancer xenografts and tumor growth monitored.Fatty nude rats were visibly obese and did not develop fasting hyperglycemia. Compared to lean rats, fatty nude rats developed fasting hyperinsulinemia, glucose intolerance, and insulin resistance. Colon cancer tumor growth rate and final weight were increased (P < 0.05) in fatty nude compared to lean rats. Final tumor weight was associated with p38 kinase phosphorylation (P < 0.01) in fatty nude rats.We have established a novel model of obesity and pre-type 2 diabetes that can be used to investigate human cancer and therapeutics in the context of obesity and its associated pathophysiology.

Antitumor Ability of KT2 Peptide Derived from Leukocyte Peptide of Crocodile Against Human HCT116 Colon Cancer Xenografts.

Many antimicrobial peptides have been shown to have anticancer activity against human cancer cell lines. Cationic KT2 peptide, derived from white blood cell extract of Crocodylus siamensis has antibacterial activity and antitumor activity against human cervical cancer cells, but there are no data on the effect of KT2 peptide on tumor growth in vivo. The anticancer activity of KT2 peptide on human colon cancer xenografts was investigated in nude mice. Tumors in nude mice (BALB/c -nu/nu mice) were induced by subcutaneous injection with HCT116 cells. Twelve days after cancer cell xenograft, mice were treated by intratumoral injection with phosphate-buffered saline or KT2 peptide (25 and 50 mg/kg) once every 2 days for a total of four times and mice were sacrificed at 2 days after the last treatment. KT2 peptide treatment did not lead to significant difference in mouse body weight among groups, but reduced both tumor volume and weight of colon cancer xenografts. Moreover, KT2 peptide increased the expression of apoptotic proteins, such as BCL2-associated X (BAX), cleaved caspase-3, and poly (ADP-ribose) polymerase and reduced that of BCL2 apoptosis regulator in xenograft tumors. This finding suggests that KT2 peptide may inhibit tumor growth via apoptosis induction in this mouse model and supports the antitumor ability of KT2 peptide.

Modulation of G2/M cell cycle arrest and apoptosis by luteolin in human colon cancer cells and xenografts.

The aim of the present study was to investigate the underlying molecular mechanisms of the potent cell cycle inhibition and apoptotic effect of luteolin on LoVo human colon cancer cells. In the present study, Cell Counting kit-8 assay revealed that luteolin exerted notable cytotoxicity on LoVo cells in a dose- and time-dependent manner, with a 50% inhibitory concentration value of 66.70 and 30.47 µmol/l at the time points of 24 and 72 h, respectively. Flow cytometric analysis confirmed that luteolin promoted cell cycle arrest at the G2/M phase, and subsequently induced cell apoptosis. Western blot analysis further revealed that luteolin exhibited an inhibitory effect on the proliferation of LoVo cells by inhibiting cell cycle arrest at the G2/M phase transition, with an inactivation of cyclin B1/cell division cycle 2 and induction of cell apoptosis, in part via cytochrome c- and deoxyadenosine triphosphate-mediated activation of apoptotic protease activating factor 1. In vivo studies revealed that luteolin effectively decreased the colon tumor body weight of mice. Therefore, the evidence suggests that luteolin may be a potential chemopreventive and chemotherapeutic agent against human colon cancer.

Antitumor activity of Lepidium latifolium and identification of the epithionitrile 1-cyano-2,3-epithiopropane as its major active component.

Consumption of Brassica (Cruciferae) vegetables is associated with a reduced risk of cancer, but identification of the active components and insights into the underlying molecular events are scarce. Here we found that an extract of Lepidium latifolium, a cruciferous plant native to southern Europe, Mediterranean countries and Asia, showed in vitro cytotoxic activity, inducing caspase-dependent apoptosis, in a variety of human tumor cells, and the plant juice showed in vivo antitumor activity in a HT-29 human colon cancer xenograft mouse model. The epithionitrile 1-cyano-2,3-epithiopropane (CETP) was identified as the major active cancer cell-killing principle of L. latifolium. Synthetic and plant-derived CETP displayed similar proapoptotic activities as assessed by biochemical and morphological analyses. Analysis of the antiproliferative capacity of CETP on a wide number of cancer cell lines from the NCI-60 cell line panel followed by COMPARE analysis, showed an activity profile different from known anticancer agents. Flow cytometry and biochemical analyses revealed that CETP-induced apoptosis involved mitochondria, as assessed by loss of mitochondrial transmembrane potential and generation of reactive oxygen species, while overexpression of Bcl-XL and Bcl-2 prevented CETP-induced apoptosis. Inhibition of reactive oxygen species by glutathione and N-acetyl cysteine reduced the apoptotic response induced by CETP. FADD dominant negative form, blocking Fas/CD95 signaling, and a specific caspase-8 inhibitor also inhibited CETP-induced killing. Taken together, our data suggest that the cancer cell-killing action of CETP, involving both intrinsic and extrinsic apoptotic signaling pathways, underlies the antitumor activity of L. latifolium plant, which could be of potential interest in cancer treatment.

SU6668 inhibits the proliferation and motility of colorectal cancer cells by inducing cycle arrest, and promotes their apoptosis

Background: The multi-targeted angiogenesis inhibitor SU6668 has demonstrated promising therapeutic effects on the progression of hematologic malignancies and certain solid tumors. Previous studies reported that SU6668 inhibited the liver metastasis of both human and mouse colon cancer xenografts via its antiangiogenic effect. This study was conducted to determine the effect of SU6668 on the proliferation, migration, and invasion of colorectal cancer cells and their rates of apoptosis.

Construction and Evaluation of a Targeted Hyaluronic Acid Nanoparticle/Photosensitizer Complex for Cancer Photodynamic Therapy.

Photodynamic therapy (PDT) is a novel treatment modality that is under intensive preclinical investigations for a variety of diseases, including cancer. Despite extensive studies in this area, selective and effective photodynamic agents that can specifically accumulate in tumors to reach a therapeutic concentration are limited. Although recent attempts have produced photosensitizers (PSs) complexed with various nanomaterials, the tedious preparation steps and poor tumor efficiency of therapy hamper their utilization. Here, we developed a CD44-targeted nanophotodynamic agent by physically encapsulating a photosensitizer, Ce6, into a hyaluronic acid nanoparticle (HANP), which was hereby denoted HANP/Ce6. Its physical features and capability for photodynamic therapy were characterized in vitro and in vivo. Systemic delivery of HANP/Ce6 resulted in its accumulation in a human colon cancer xenograft model. The tumor/muscle ratio reached 3.47 ± 0.46 at 4 h post injection, as confirmed by fluorescence imaging. Tumor growth after HANP/Ce6 treatment with laser irradiation (0.15 W/cm2, 630 nm) was significantly inhibited by 9.61 ± 1.09-fold compared to that in tumor control groups, which showed no change in tumor growth. No apparent systemic and local toxic effects on the mice were observed. HANP/Ce6-mediated tumor growth inhibition was accessed and observed for the first time by 18F-fluoro-2-deoxy-d-glucose positron emission tomography as early as 1 day after treatment and persisted for 14 days within our treatment time window. In sum, our results highlight the imaging properties and therapeutic effects of the novel HANP/Ce6 theranostic nanoparticle for CD44-targeted PDT cancer therapy that may be potentially utilized in the clinic. This HANP system may also be applied for the delivery of other hydrophobic PSs, particularly those that could not be chemically modified.

Inhibitory effect of bcl-XL antisense oligodeoxynucleotides on the proliferation of colon cancer cells and the growth of human colon cancer xenografts in nude mice

Objective To investigate the effect of bcl-XL antisense oligonucleotide transfection on the proliferation of colon cancer cells and the growth of human colon cancer xenografts in nude mice. Methods A long-term SW620 single cell suspension was prepared for subcutaneous injection in nude mice. Nude mice were randomly divided into 3 groups: control group, N-ODN group and antisense oligonucleotide group (n=5), with an average of 50 mm3. The antisense oligonucleotide group and N-ODN group were injected with 2 ml/kg antisense oligonucleotide or N-ODN, and the control group was injected with equal amount of normal saline. At 16th day after the operation, the tumor cells were taken out, and some tumor tissues were extracted. RNA and protein were extracted. The cationic liposome mediated transfection of antisense oligonucleotide, real-time quantitative polymerase chain reaction (Real-time PCR), Western blotting, methyl thiazol tetrazolium (MTT) method and TdT-mediated dUTP nick end labeling (TUNEL) method were applied to observe the effects of bcl-XL antisense oligonucleotide on the proliferation and apoptosis of colorectal cancer cells in vivo and the growth of human colon cancer xenografts. Results (1) The inhibition rate of cell proliferation activity was gradually increased with the increase of antisense oligonucleotide concentration. When the concentration was over 200 nmol/L, the inhibition rate of cell proliferation was significantly decreased with the difference being statistically significant (t=2.418, 1.531 and 1.838, P=0.003, 0.016 and 0.011). (2) The relative expression levels in the control group, blank control group, N-ODN group and mRNA antisense oligonucleotides group were 0.96±0.25, 0.97±0.48, 0.78±0.32 and 0.41±0.13, the inhibition rate was 0.00%, -6.25%, 18.75% and 57.29%, respectively. There was significant difference between antisense oligonucleotide group and control group, blank control group or N-ODN group (P=0.025). (3) Compared with the control group, there was no significant change in the blank control group; the N-ODN group was slightly lower than that in the control group; the antisense oligonucleotide group was significantly lower (P=0.017). (4) The positive rates of cells in control group, blank control group, N-ODN group and antisense oligonucleotide group were 3.7%, 5.0%, 7.0% and 35.0%, respectively. By statistical analysis, antisense oligonucleotide group and cell control group, blank control group and N-ODN group, the difference was statistically significant (χ2=181.880, P=0.000); cells in the control group, N-ODN group and control group comparison between 22 blank, there was no statistically significant difference (χ2=1.410, P=0.084). (5) There was no significant difference in tumor volume between first groups (P=0.162). On the sixth day, there was significant difference between the antisense oligonucleotide group and the control group (t=2.410, P=0.027). On the eleventh and the 16 day, there was significant difference between the two groups in (P=0.028, 0.016). (6) The expression of bcl-XL gene in the tumor tissues of the 3 groups of nude mice was significantly decreased after antisense oligonucleotide treatment, but the expression of antisense oligonucleotide group was significantly decreased, while the expression of N-ODN in the control group and the control group did not change significantly. Conclusion bcl-XL antisense oligonucleotides can effectively inhibit the proliferation of colon cancer cells and the growth of human colon cancer xenografts in nude mice. To reduce the expression of bcl-XL by antisense oligonucleotides, and to provide the experimental basis for gene therapy of colon cancer. Key words: Colon cancer; Oligonucleotides; Bcl-XL; Proliferation

Abstract 4042: N-121, a novel polypeptide conjugate of SN-38, demonstrated enhanced intratumoral accumulation and sustained release of free SN-38 with potent antitumor activity

Abstract Introduction: SN-38 is a critical chemotherapeutic agent active against a variety of solid tumors including advanced colorectal cancer. Clinical use of SN-38 is hampered by its poor solubility in pharmaceutically acceptable solvents. Although irinotecan, the only approved prodrug of SN-38, is widely used, its efficacy is limited by low conversion to the active SN-38 as well as hematologic and gastrointestinal dose-limiting toxicities. In an attempt to improve the water solubility, antitumor activity and safety profiles of SN-38, we synthesized a water soluble polypeptide-SN-38 conjugate (N-121) and examined its antitumor activity and pharmacokinetics (PK) in mouse models. Methods: Antitumor activity and PK of SN-38, administered as N-121 or irinotecan, was evaluated following tail vein injection in male nude mice with subcutaneous orthotopic xenografts of the HCT116 human colon cancer. Mice received N-121 400 mgk (mg/kg) (32 mkg of eq. SN-38) or irinotecan 80 mgk once a week for 2 weeks and were followed for up to 46 days. Results: N-121 drastically improved by over 1000 fold the water solubility of SN-38. At a SN-38 equivalent dose of less than half of that of irinotecan, N-121 resulted in a much longer tumor growth delay (36 days) than irinotecan (14 days). The more durable tumor growth inhibition with N-121 was consistent with its enhanced intratumoral accumulation: intratumoral trough levels of SN-38 were about 250-fold higher than those observed in plasma, compared with only 15-fold with irinotecan. With N-121, Intratumoral levels of SN-38 remained stable over the PK observation period (48 h), contrasting to a marked decline in SN-38 levels with irinotecan; intratumoral trough levels of SN-38 from N-121 were about 50-fold higher than those achieved with irinotecan. N-121 was well tolerated with a slight weight loss of 7.2%. Conclusion: N-121, a novel water soluble polypeptide-SN-38 conjugate, exhibited enhanced and sustained intratumoral exposure of SN-38 with high antitumor activity in a mouse xenograft tumor model. These promising results warrant further evaluation of N-121. Note: This abstract was not presented at the meeting. Citation Format: Haojun Wang, Wei Li, Xiaojian Zhou, Liuyi Wan, Guanghui Wen. N-121, a novel polypeptide conjugate of SN-38, demonstrated enhanced intratumoral accumulation and sustained release of free SN-38 with potent antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4042. doi:10.1158/1538-7445.AM2017-4042

Sphingosine kinase 1 expression enhances colon tumor growth

BackgroundAccumulating evidence suggests that sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate pathway plays a pivotal role in colon carcinogenesis.MethodsTo further support the evidence, we investigated the effects of SphK1 using three separate animal models: SphK1 knockout mice, SphK1 overexpressing transgenic mice, and SphK1 overexpression in human colon cancer xenografts. Using azoxymethane (AOM, colon carcinogen), we analyzed colon tumor development in SphK1 KO and SphK1 overexpression in intestinal epithelial cells regulated by a tet-on system. Then, we analyzed subcutaneous tumor growth using xenografts of HT-29 human colon cancer cell. Finally, immunohistochemical analyses for SphK1 and COX-2 were performed on human colon cancer tissue microarray.ResultsSphK1 KO mice, compared to wild-type mice, demonstrated a significant inhibition in colon cancer development induced by AOM (58.6% vs. 96.4%, respectively, P < 0.005). Tumor multiplicity (1.00 vs. 1.64 per colon, respectively, P < 0.05) and tumor volume (14.82 mm3 vs. 29.10 mm3, P < 0.05) were both significantly reduced in SphK1 KO mice compared to wild-type mice. Next, SphK1 overexpression in HT-29 enhanced tumor growth as compared to GFP control in nude mice (229.5 mm3 vs. 90.9 mm3, respectively, P < 0.05). Furthermore, overexpression of SphK1 in intestinal epithelial cells significantly enhances AOM-induced colon tumor formation (P < 0.05). Lastly, SphK1 and COX-2 intensity tended to reduce overall survival of late stage colon cancer patients.ConclusionsSphK1 expression regulates the early stage of colon carcinogenesis and tumor growth, thus inhibition of SphK1 may be an effective strategy for colon cancer chemoprevention.

Coptisine from Rhizoma Coptidis Suppresses HCT-116 Cells-related Tumor Growth in vitro and in vivo

Colorectal cancer is one of the most common causes of cancer-related death in humans. Coptisine (COP) is a natural alkaloid from Coptidis Rhizoma with unclear antitumor mechanism. Human colon cancer cells (HCT-116) and xenograft mice were used to systematically explore the anti-tumor activity of COP in this study. The results indicated that COP exhibited remarkably cytotoxic activities against the HCT-116 cells by inducing G1-phase cell cycle arrest and increasing apoptosis, and preferentially inhibited the survival pathway and induced the activation of caspase proteases family of HCT-116 cells. Experimental results on male BALB/c nude mice confirmed that orally administration of COP at high-dose (150 mg/kg) could suppress tumor growth, and may reduce cancer metastasis risk by inhibiting the RAS-ERK pathway in vivo. Taken together, the results suggested that COP may be potential as a novel anti-tumor candidate in the HCT-116 cells-related colon cancer, further studies are still needed to suggest COP for the further use.