Efficacy Of Chemopreventive Agents Research Articles

Camel milk-derived exosomes as novel nanocarriers for curcumin delivery in lung cancer

Cancer remains a leading cause of mortality, with non-small cell lung cancer (NSCLC) being a primary contributor to cancer-related deaths. Traditional treatment strategies such as chemotherapy, radiation, and hormone therapy often present challenges, including severe side effects, drug resistance, and toxicity. Recent advancements in nanotechnology aim to enhance the effectiveness of cancer therapies by targeting drugs selectively and specifically to tumor cells. Among these innovations, exosomes, or small extracellular vesicles (EVs), have emerged as promising carriers for drug delivery due to their natural origin and ability to encapsulate both small molecules and biologics. This study explores the use of exosomes derived from camel milk in Hail, Saudi Arabia, as a vehicle for delivering curcumin (CUR), a polyphenol with known chemopreventive properties but limited bioavailability. Camel milk was processed to isolate exosomes through differential centrifugation, followed by characterization using dynamic light scattering, zeta potential measurements, and Western blot analysis to confirm exosomal markers. The encapsulation of CUR into camel milk-derived exosomes demonstrated a 20% loading efficiency as analyzed by UPLC. In vitro antiproliferative assays revealed that the exosomal formulation of CUR (ExoCUR) significantly enhanced cytotoxicity against drug -sensitive (A549) and taxol-resistant (A549TR) lung cancer cells compared to free CUR. Molecular docking studies and molecular dynamics simulations indicated that CUR has a strong binding affinity for the epidermal growth factor receptor (EGFR), comparable to the established drug gefitinib. Furthermore, CUR effectively downregulated EGFR and STAT3 expression in cancer cells, suggesting its potential to disrupt key signaling pathways involved in tumor progression. Our findings highlight the potential of camel milk-derived exosomes as an effective and biocompatible delivery system for CUR, offering a promising strategy to overcome the limitations of current cancer therapies and enhance the therapeutic efficacy of chemopreventive agents.

Natural Products as Cancer Chemo Preventive Agents: Where We Stand

This work briefly reviews cancer chemoprevention. This is a very challenging field, as products with a high level of toxicity such as chemotherapeutic agents may be proposed and accepted only under life-threatening conditions. Cancer chemoprevention is otherwise limited to completely safe substances, preferably having neither toxic nor side effects, administered in relatively low amounts. Phases of clinical trials, therapeutic end-points, and biomarkers of chemoprevention are difficult to be defined. The clinical trials needed to prove the efficacy of chemopreventive agents must be very long and extremely widespread to achieve significance, with many variables difficult to control, and therefore subjected to many confounding factors. This makes them almost impossible. It is, therefore, no surprise, if the progress of chemoprevention has been so far very limited. There are only a few examples of direct use of chemopreventive agents, under investigation, but with anything but established protocols, in addition to indirect uses such as general supplementation with antioxidant, anti-inflammatory, and immune-supportive agents. Cancer chemoprevention remains a potentially very rewarding approach, certainly worth further study, but extremely difficult to pursue, in need of different methodological approaches to producing valuable chemopreventive compounds of clear dosages and benefits.

Use of Biomarker Modulation in Normal Mammary Epithelium as a Correlate for Efficacy of Chemopreventive Agents Against Chemically Induced Cancers.

In both estrogen receptor/progesterone receptor-positive (ER+/PR+) human breast cancer and in ER+/PR+ cancers in the methylnitrosourea (MNU)-induced rat model, short-term modulation of proliferation in early cancers predicts preventive/therapeutic efficacy. We determined the effects of known effective/ineffective chemopreventive agents on proliferative index (PI) in both rat mammary epithelium and small cancers. Female Sprague-Dawley rats were treated with MNU at 50 days of age. Five days later, the rats were treated with the individual compounds for a period of 14 days. At that time, normal mammary tissue from the inguinal gland area was surgically removed. After removal, the rats remained on the agents for an additional 5 months. This cancer prevention study confirmed our prior results of striking efficacy with tamoxifen, vorozole, Targretin, and gefitinib, and no efficacy with metformin, naproxen, and Lipitor. Employing a separate group of rats, the effects of short-term (7 days) drug exposure on small palpable cancers were examined. The PI in both small mammary cancers and in normal epithelium from control rats was >12%. In agreement with the cancer multiplicity data, tamoxifen, vorozole, gefitinib, and Targretin all strongly inhibited proliferation (>65%; P < 0.025) in the normal mammary epithelium. The ineffective agents metformin, naproxen, and Lipitor minimally affected PI. In the small cancers, tamoxifen, vorozole, and Targretin all reduced the PI, while metformin and Lipitor failed to do so. Thus, short-term changes in the PI in either normal mammary epithelium or small cancers correlated with long-term preventive efficacy in the MNU-induced rat model.

Efficacy of Chemopreventive Agents on Disease-free and Overall Survival in Patients With Pancreatic Ductal Adenocarcinoma: The CAOS Study

Efficacy of Chemopreventive Agents on Disease-free and Overall Survival in Patients With Pancreatic Ductal Adenocarcinoma: The CAOS Study

Nanoformulation of EPZ011989 Attenuates EZH2–c-Myb Epigenetic Interaction by Proteasomal Degradation in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a malignant disorder of hematopoietic progenitor cells with a poor prognosis of 26% of patients surviving 5 years after diagnosis. Poor bioavailability and solubility are significant factors limiting the efficacy of chemopreventive agents. In AML, the epigenetic regulator polycomb group of protein member EZH2 is highly expressed and is essential for the survival of leukemic cells. An EZH2-specific inhibitor, EPZ011989, encapsulated in human serum albumin nanoparticles (HSANPs) was synthesized for the first time via the desolvation method. The noncovalent interactions between EPZ011989 and HSANPs in nanocomposites facilitating the efficient loading and sustainable release of the drug showed enhanced cellular uptake and nuclear localization of EPZ011989-loaded HSANPs in human AML cell lines. The reduction of cell viability, colony formation inhibition, cell cycle arrest at the G2/M phase, and cell proliferation assay promoting apoptosis through the loss of mitochondrial homeostasis exerting antileukemic activity were evident. The real-time polymerase chain reaction (PCR) and western blot-based studies showed that the present nanoformulation reduces the level of PcG proteins, including EZH2, BMI-1, etc. This downregulation is associated with reduced H3K27me3 and H2AK119ub modifications conferring chromatin compaction. The immunoprecipitation study showed the physical interaction of EZH2 and c-Myb can be linked to the regulation of leukemogenesis. Further investigation revealed the mechanism of EZH2 and c-Myb downregulation via ubiquitination and proteasomal degradation pathway, confirmed by using proteasome inhibitor, suggesting the key role of proteasomal degradation machinery. Moreover, c-Myb interacted with the EZH2 promoter, which is evident by the chromatin immunoprecipitation assay and siRNA silencing. Furthermore, the formulation of EPZ011989 in HSANPs improved its biodistribution in vivo and showed excellent aqueous dispersibility and biocompatibility. In vivo studies further showed that EPZ011989-loaded HSANPs reduce the expression of CD11b+ and CD45+ markers in immunophenotyping from peripheral blood and bone marrow in engrafted nude mice. Targeted depletion of EZH2 alleviated the disease progression in nude mice and prolonged their survival. The findings provide valuable experimental evidence for the targeted epigenetic therapy of AML. The present results demonstrate an epigenetic regulation-based superior antileukemic therapy.

A novel and cost-effective ex vivo orthotopic model for the study of human breast cancer in mouse mammary gland organ culture.

ABSTRACTMouse mammary organ culture (MMOC) is used to evaluate the efficacy of chemopreventive agents against the development of carcinogen-induced preneoplastic lesions and is highly correlative to in vivo carcinogenesis models. Here, we developed a new ex vivo MMOC model, by introducing human breast cancer cells into the mouse mammary gland. This novel model, termed human breast cancer in MMOC (BCa-MMOC), mimics in vivo orthotopic breast cancer mouse models. To develop this model, estradiol- and progesterone-sensitized female mice were injected with letrozole-sensitive and -resistant T47D breast cancer cells in the mammary glands and then euthanized. The glands were cultured in vitro with hormone-supplemented media. On day 25, the glands were fixed and processed by histopathology and immunohistochemistry to evaluate for the presence of T47D cells, growth pattern, cancer markers and estradiol responsiveness. Histopathological analyses demonstrated an identical pattern of growth between the breast cancer cells injected ex vivo and in vivo. Interestingly, clusters of cancer cells in the mammary gland stroma appeared similar to those observed in human breast tumors. The injected T47D cells survived and proliferated for 15 days maintaining expression of estrogen receptor alpha (ER), progesterone receptor (PR), epidermal growth factor receptor (EGFR), and aromatase. The aromatase-overexpressing T47D grown in the BCa-MMOC sufficiently metabolized estrogen, resulting in enhanced cell proliferation, induction of estrogen target genes (i.e. ER and PR-B), and showed typical changes to estrogenic milieu. In summary, here we show a novel, inexpensive ex vivo model, to potentially study the effects of therapeutic agents on cancer cells grown in an orthotopic micromilieu.This article has an associated First Person interview with the first author of the paper.

Abstract 5088: Silibinin inhibits UVB-induced promotion and progression of microscopic basal cell carcinoma formation via targeting bone morphogenic protein 2

Abstract Non-melanoma skin cancers (NMSCs) account for about half of all malignancies diagnosed annually in the United States. Around 80% of NMSCs are basal cell carcinoma (BCC) and 20% are squamous cell carcinoma (SCC). Whereas the efficacy of several chemopreventive agents has been examined and reported against both BCC and SCC, a majority of these studies have focused on the test agent’s activity in a long-term setting to determine the amount of tumors formed. Notably, the studies evaluating the efficacy of chemopreventive agents during early stage/s of BCC development are lacking. Accordingly, utilizing the well-established patched (Ptch)+/- mouse model of UVB-induced BCC formation, we excised skin samples from UVB exposed mice prior to tumor formation to study the promotion/progression of BCC and to determine the target/s of silibinin, a well-known skin cancer (SCC) chemopreventive agent, in tumor growth inhibition. Ptch+/- and Ptch+/+ mice were irradiated with UVB 240mJ/cm2 3 times per week with or without topically applied silibinin 5 times per week. As early as one month, we found that UVB exposure significantly increased the number of mast cells in Ptch+/- mice by about 48% (P&amp;lt;0.05), which was completely inhibited (to control levels) by silibinin topical treatments. In Ptch+/+ mice, which do not develop BCC tumors, we did not observe any increase in mast cells following UVB exposure, suggesting this could be a specific pathway in the development of BCC. To decipher the molecular mechanism of these findings, we performed a PCR profiler array analysis of several genes involved in signal transduction pathways which showed strong differences between Ptch+/+ and Ptch+/- mice that were unexposed, UVB irradiated, and silibinin treated. Most notably, following UVB exposure for 1 month, in Ptch+/- mice the expression of Bone morphogenetic protein 2 (BMP-2), Hairy/enhancer-of-split related with YRPW motif 1 (Hey1), and Inhibitor of DNA binding 1 (Id1) was significantly upregulated when compared to Ptch+/+ mice. Additional studies focusing on BMP-2 found that silibinin strongly inhibits UVB-induced expression of BMP-2 in Ptch+/- mouse skin. Consistent with these results, we also found that silibinin strongly attenuates UVB-induced BMP-2 expression and DNA damage in Ptch+/- mouse skin ex vivo. Regarding BCC formation, silibinin treatment inhibited UVB-induced microscopic BCC formation in Ptch+/- mice; microscopic tumor number and size were reduced by 73% and 84%, respectively. Together, our results suggest a possible role of BMP-2 in early stages of BCC development and that silibinin plausibly acts through BMP-2 to inhibit microscopic BCC formation. In conclusion, our previous studies in SCC models and current findings in BCC model suggest that silibinin could be a multi-target agent capable of being a chemopreventive agent for both types of NMSCs. Citation Format: Cynthia M. Rigby, Gagan Deep, Anil K. Jain, David J. Orlicky, Chapla Agarwal, Komal Raina, Rajesh Agarwal. Silibinin inhibits UVB-induced promotion and progression of microscopic basal cell carcinoma formation via targeting bone morphogenic protein 2 [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 5088.

Silibinin inhibits ultraviolet B radiation-induced mast cells recruitment and bone morphogenetic protein 2 expression in the skin at early stages in Ptch(+/-) mouse model of basal cell carcinoma.

Around 80% of nonmelanoma skin cancers (NMSCs) are basal cell carcinoma (BCC), still studies evaluating the efficacy of chemopreventive agents during early stage/s of BCC development are lacking. Accordingly, utilizing the well-established patched (Ptch)+/- mouse model of ultraviolet B (UVB) radiation-induced BCC formation, we excised skin samples from UVB exposed Ptch+/- and Ptch+/+ mice before tumor formation to study the promotion/progression of BCC and to determine the efficacy and target/s of silibinin, a well-known skin cancer chemopreventive agent. UVB exposure for 1 month increased the number of mast cells in Ptch+/- mice by ~48% (P < 0.05), which was completely inhibited by silibinin. Polymerase chain reaction profiler array analysis of skin samples showed strong molecular differences between Ptch+/+ and Ptch+/- mice which were either unexposed or UVB irradiated+/- silibinin treatment. Most notably, silibinin treatment significant decreased the expression of BMP-2, Bbc3, PUMA, and Ccnd1 in Ptch+/- mice irradiated with silibinin + UVB. Additional studies showed that silibinin targets UVB-induced expression of bone morphogenetic protein 2 (BMP-2) in Ptch+/- mouse skin. Last, our studies found that silibinin strongly attenuates UVB-induced BMP-2 expression and DNA damage in Ptch+/- mouse skin ex vivo only after single UVB exposure. Together, our results suggest a possible role of mast cell recruitment and BMP-2 activation in the early stages of BCC development; these are strongly inhibited by silibinin suggesting its possible chemopreventive efficacy against BCC formation in long-term UVB exposure regimen.

Chemoprevention of prostate cancer in men with high-grade prostatic intraepithelial neoplasia (HGPIN): a systematic review and adjusted indirect treatment comparison.

BackgroundHigh-grade prostatic intraepithelial neoplasia (HGPIN) is the precursor or premalignant form of prostate cancer. At least 30% patients with a confirmed HGPIN will develop prostate cancer within 1 year after repeated biopsy. HGPIN patients are the appropriate at-risk population for chemoprevention strategies investigation against prostate cancer. However the commonly used chemoprevention agents that targeted on hormonal imbalance or lifestyle-related factors showed varied results in HGPIN patients.MethodsLiterature searches were conducted in PubMed, EMBASE and Cochrane library according to Cochrane guidelines before January 31st, 2017. Direct meta-analysis were performed to summarize the efficacy of candidate chemopreventative agents Dutasteride, Flutamide, Toremifene, Selenium, Green tea components, Lycopene and natural food products combination. Adjusted indirect meta-analyses were employed to compare the relative efficacy of these candidate chemoprevention agents head-to-head.ResultsThe overall incidence of prostate cancer in HGPIN was slightly decreased by chemoprevention agents (25.7% vs 31.5%, RR = 0.92, 95% CI: 0.83-1.03, P = 0.183), with minor heterogeneity (I2 = 22.3%, χ2 = 15.08, P = 0.237), but without statistical significance. Subgroup analysis showed that green tea catechins significantly decreased prostate cancer in HGPIN patients (7.60% vs 23.1%, RR = 0.39, 95% CI: 0.16-10.97, P P = 0.044), with moderate heterogeneity (I2 = 47.9%, χ2 = 1.92, P = 0.166). The adjusted indirect meta-analysis favored green tea catechins over other chemoprevention agents, and significantly when compared to natural food products combination (RR = 0.355, 95% CI: 0.134-0.934).ConclusionThe overall efficacy of chemoprevention agents in HGPIN patients is limited. But Green tea catechins showed the superiority to decrease prostate cancer in HGPIN patients.

Abstract B53: Predicting efficacy of chemopreventive agents in animal tumor assays by statistical modeling

Abstract NCI's Division of Cancer Prevention has over 25 years entered some 800 agents into a program designed to test cancer preventive efficacy. Early in the testing pathway are 2 sequential critical steps: (1) in vitro/in vivo morphologic assays and, (2) for agents successful in step 1, cancer-preventive testing (measured in terms of tumor incidence and multiplicity reduction) in animal tumor assays. The ultimate intention is to follow successful testing in animals with entry into clinical trial testing in humans. To facilitate the optimal selection of appropriate morphologic tests for specific disease-site animal tumor models, we undertook the current project. We evaluated the success of our strategy by determining how accurately the earlier stage (morphologic) assays predict efficacy in the later-stage (animal tumor) assays. Focusing on 210 agents that were tested in both morphologic and animal tumor assays, we carried out statistical modeling of how well the 6 most commonly used morphologic assays predicted drug efficacy in animal tumor models. Using multimodel inference, three statistical models were generated to evaluate the ability of these 6 morphologic assays to predict tumor outcomes in 3 different sets of animal tumor assays: (1) all tumor types, (2) breast cancer only, and (3) colon cancer only. Using this statistical modeling approach, each morphologic assay was assigned a value reflecting how strongly it predicted outcomes in each of the 3 different sets of animal tumor assays. The goal is to use predictive models such as these to guide our future decision-making with respect to selection of preventive agents as well as morphologic and animal tumor assays. In this manner, we hope to improve the efficiency of our overall approach to chemoprevention agent development. Citation Format: Barbara K. Dunn, Vernon E. Steele, Richard M. Fagerstrom, Carol F. Topp, David Ransohoff, Leslie G. Ford, Barnett S. Kramer. Predicting efficacy of chemopreventive agents in animal tumor assays by statistical modeling. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B53.

Nanoparticles increase the efficacy of cancer chemopreventive agents in cells exposed to cigarette smoke condensate.

Lung cancer is a leading cause of death in developed countries. Although smoking cessation is a primary strategy for preventing lung cancer, former smokers remain at high risk of cancer. Accordingly, there is a need to increase the efficacy of lung cancer prevention. Poor bioavailability is the main factor limiting the efficacy of chemopreventive agents. The aim of this study was to increase the efficacy of cancer chemopreventive agents by using lipid nanoparticles (NPs) as a carrier. This study evaluated the ability of lipid NPs to modify the pharmacodynamics of chemopreventive agents including N-acetyl-L-cysteine, phenethyl isothiocyanate and resveratrol (RES). The chemopreventive efficacy of these drugs was determined by evaluating their abilities to counteract cytotoxic damage (DNA fragmentation) induced by cigarette smoke condensate (CSC) and to activate protective apoptosis (annexin-V cytofluorimetric staining) in bronchial epithelial cells both in vitro and in ex vivo experiment in mice. NPs decreased the toxicity of RES and increased its ability to counteract CSC cytotoxicity. NPs significantly increased the ability of phenethyl isothiocyanate to attenuate CSC-induced DNA fragmentation at the highest tested dose. In contrast, this potentiating effect was observed at all tested doses of RES, NPs dramatically increasing RES-induced apoptosis in CSC-treated cells. These results provide evidence that NPs are highly effective at increasing the efficacy of lipophilic drugs (RES) but are not effective towards hydrophilic agents (N-acetyl-L-cysteine), which already possess remarkable bioavailability. Intermediate effects were observed for phenethyl isothiocyanate. These findings are relevant to the identification of cancer chemopreventive agents that would benefit from lipid NP delivery.

Efficacy of chemopreventive agent, Dipteracanthus prostratus, on 1,2-dimethylhydrazine induced colon carcinogenesis

Aim: The aim of this study was to evaluate the effect of Dipteracanthus prostratus extracts on 1,2-dimethyl hydrazine (DMH) induced colon cancer in rats. Materials and Methods: To achieve this objective DMH model was selected to evaluate the effect of D. prostratus extracts on colon cancer. The study was carried out on 48 male Wistar rats divided into six groups. Group I served as a control. Groups II and III served as a positive control received ethanolic extract (500 mg/kg) and aqueous extract (500 mg/kg), respectively. Group IV served as a tumor bearing group received (20 mg/kg) DMH. Groups V and VI served as a treatment group received DMH (20 mg/kg) + ethanolic extract (500 mg/kg) and aqueous extract (500 mg/kg), respectively. Lipid peroxidation (LPO) was studied by measuring the formation of thiobarbituric acid reactive substances, catalase (CAT), glutathione peroxidase (GP X ), and reduced glutathione (GSH) in the liver and colonic tissues of DMH administered rats. Results: (1) Decreased levels of LPO in the colonic tissues; (2) decreased activities of antioxidant enzymes LPO, CAT, GP X , and GSH levels in the tissues on DMH treatment. D. prostratus supplementation during the entire period stages of carcinogenesis significantly reversed these activities. Conclusion: These results indicate that D. prostratus may be a potential chemopreventive agent against DMH induced colon cancer.

Abstract 238: Vitamin D sufficiency slows the progression of dysplasic lesions in the NTCU mouse model of lung squamous cell carcinoma

Abstract Progress has recently been made in identifying populations at risk for lung cancer using genetic, clinical and demographic information. The N-nitroso-tris-chloroethylurea (NTCU) mouse model, in which animals develop premalignant histopathology similar to that seen in humans, is used to examine the potential efficacy of chemoprevention agents to be utilized in at risk populations. We identified 25 µl of 40 mM NTCU /week as the optimal dosing regimen in SWR/J mice for chemoprevention studies. At this dose, topical treatments of NTCU induce predominantly low-grade dysplastic lesions by 15 weeks (w) and high-grade dysplastic (HGD) lesions by 25 w in the large airways. Additionally we found that NTCU stimulates a state of chronic inflammation associated with the development of dysplasias. Epidemiologic studies indicate that there is an inverse relationship between vitamin D and the risk and prognosis of lung cancer. Vitamin D acts through the vitamin D receptor to promote cellular differentiation and inhibit proliferation and inflammation. The effect of vitamin D on cancer progression was tested in the NTCU model, using dietary vitamin D3 (0 or 2000 IU/kg) alone and in combination with intraperitoneal injections of the active metabolite of vitamin D, calcitriol (80 ug/kg). Female mice were randomized to 6 treatment groups (n=15 mice/group/time point (15 and 25 w)). Disease was evaluated by enumerating the percentage of HGD lesions, per the total area in serial H&amp;E sections of the lung. The percentage of HGD lesions in the large airways was reduced in the vitamin D sufficient mice (SN) (8.72%, P&amp;lt;0.05), SN + calcitriol (SN+C) (6.59%, P&amp;lt;0.05) and the vitamin D deficient + calcitriol (DN+C) (12.3% P&amp;lt;0.05) groups compared to the DN (22.67%) group after 15 w of NTCU. The percentage of HGD lesions in the SN groups remained significantly less than the DN (35.5%) and DN+C (21.3%) groups, with little increase in the SN (8.43% P&amp;lt;0.05) and SN+C (11.81% not significant (NS)) groups after 25 w. Furthermore, there was a significant increase in proliferation associated with increased HGD. Following 25 w of NTCU treatment there was a 2-fold increase in Ki-67 staining all groups compared all groups after 15 w of NTCU. However, the was 30% more Ki-67staining in the DN (P&amp;lt;0.001) group and addition of calcitriol reduced proliferation by 12% and 6% in the DN+C (P&amp;lt;0.01) and SN+C (NS) groups. Moreover, vitamin D deficiency was associated with an increased systemic and local inflammation marked by a 3-fold increase in circulating white blood cells (WBCs) (P&amp;lt;0.05), a 20% increase in IL-6 levels (P&amp;lt;0.05) and a 4 -fold increase in WBCs in bronchial lavages (P&amp;lt; 0.05) in the DN group. In conclusion this study indicates that vitamin D deficiency promotes the development of dysplasia, increases proliferation and inflammation, which is likely promote the development of frank squamous cell carcinoma. Supported by NIH/NCCAM F31AT0006487 (Mazzilli) NIH/NCI CA067267 (Johnson) Citation Format: Sarah A. Mazzilli, Mary E. Reid, Paul N. Bogner, Kristopher Attwood, Pamela A. Hershberger, Donald L. Trump, Candace S. Johnson. Vitamin D sufficiency slows the progression of dysplasic lesions in the NTCU mouse model of lung squamous cell carcinoma. [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 238. doi:10.1158/1538-7445.AM2014-238

Chemoprevention of Colon and Small Intestinal Tumorigenesis in APCmin/+ Mice By SHetA2 (NSC721689) without Toxicity

The occurrence of intestinal polyps in people at high risk for developing colorectal cancer provides an opportunity to test the efficacy of chemoprevention agents. In this situation of treating otherwise healthy people, the potential for toxicity must be minimal. The small-molecule flexible heteroarotinoid (Flex-Het), called SHetA2, has chemoprevention activity in organotypic cultures in vitro and lack of toxicity at doses capable of inhibiting xenograft tumor growth in vivo. The objective of this study was to evaluate SHetA2 chemoprevention activity and toxicity in the APC(min/+) murine model. Oral administration of SHetA2 at 30 and 60 mg/kg five days per week for 12 weeks significantly reduced development of intestinal polyps by 40% to 60% depending on the dose and sex of the treatment group. Immunohistochemical and Western blot analysis of polyps showed reduced levels of cyclin D1 and proliferating cell nuclear antigen in both SHetA2 treatment groups. Western blot analysis also showed SHetA2 induction of E-cadherin, Bax, and caspase-3 cleavage along with reduction in Bcl-2, COX-2, and VEGF, consistent with SHetA2 regulation of apoptosis, inflammation, and angiogenesis. Neither dose caused weight loss nor gross toxicity in APC(min/+) or wild-type littermates. Magnetic resonance imaging (MRI) of cardiac function showed no evidence of SHetA2 toxicity. SHetA2 did not alter left ventricular wall thickness. In summary, SHetA2 exerts chemoprevention activity without overt or cardiac toxicity in the APC(min/+) model. SHetA2 modulation of biomarkers in colon polyps identifies potential pharmacodynamic endpoints for SHetA2 clinical trials.

Abstract 2486: Plasma proteomic changes induced by effective chemopreventive ratios of N-3:N-6 fatty acids against MNU-induced mammary carcinogenesis of the rat.

Abstract The development of sensitive, specific, and reliable biomarkers for early detection of breast cancer and the discovery of novel chemopreventive agents are two strategies with promising clinical applications. It has been postulated that the risk of breast cancer can be influenced by certain dietary compounds such as the type and amount of dietary fats ingested. We have shown that a diet high in an n-3:n-6 ratio (10:1 and 25:1), that provided 30% calories from fat (10% monounsaturated, 10% polyunsaturated, and 10% saturated fatty acids), significantly reduced rat mammary cancer induced by 1-methyl-1-nitrosourea(MNU). In the present study we used a proteomic approach to gain insights into the mechanisms of protection by a high n-3:n-6 ratio with the goal of identifying biomarkers that can be employed to monitor efficacy of chemopreventive agents. The plasma from six rats from each of three groups, a control group(n-3:n-6=1:1) and two groups fed a high n-3 diet(n-3:n-6=10:1 and 25:1) was pooled and submitted to the isobaric Tags for Relative and Absolute Quantitation(iTRAQ) method, which includes matrix-assisted laser desorption/ionization tandem mass spectroscopy(MALDITOF/TOF) to identify and quantify proteins that were regulated as a result of n-3 fatty acid supplementation. There were 148 proteins identified in our study with 95% confidence(unused score 1.3 or greater) by ProteinPilot 4.0. In plasma of rats fed 10:1 n-3:n-6, and 25:1 n-3:n-6 the number of proteins that met our criteria(p&amp;lt;0.05, error factor≤2) were 10 and 14 proteins, respectively. Our results illustrated that many proteins were modified in a manner consistent with chemoprevention. Validation of the iTRAQ by Western Blotting showed up-regulation of vitamin D binding protein and gelsolin postulated to have a tumor suppressive effect through modulation of inflammation and actin depolarization, respectively. We also observed that 14-3-3 sigma, a well known tumor suppressor in breast tissue, was increased in a ratio dependent fashion. In contrast, we observed down-regulation of alpha-1b-glycoprotein which has been reported to be elevated in the serum of breast cancer patients. These proteins may serve in future studies as candidate biomarkers in early detection and clinical chemoprevention trials with well-defined ratios of n-3:n-6 fatty acids. Support: Susan G. Komen Foundation(KG081632) Citation Format: Christine G. Skibinski, Henry J. Thompson, Arunangshu Das, Andrea Manni, Karam El-Bayoumy. Plasma proteomic changes induced by effective chemopreventive ratios of N-3:N-6 fatty acids against MNU-induced mammary carcinogenesis of the rat. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2486. doi:10.1158/1538-7445.AM2013-2486

Molecular medicine and the development of cancer chemopreventive agents

Chemoprevention is effective in inhibiting the onset of cancer in experimental animal models, but the transferability of similar results to humans is questionable. Therefore, reliable intermediate molecular biomarkers are needed to evaluate the efficacy of chemopreventive agents before the onset of cancer. The use of genomic biomarkers is limited by their poor predictive value. Although post-genomic biomarkers (i.e., gene-expression analyses) are useful for evaluating the safety, efficacy, and mechanistic basis of chemopreventive agents, the biomarkers are often poorly related to the phenotype, due to posttranscriptional regulation. Proteome analyses can evaluate preclinical phenotype alterations, but only at low protein counts. MicroRNA alterations, which are essential for the development of cancer, may be modulated by chemopreventive agents. Furthermore, microRNA delivery may be used to counteract carcinogenesis. Exposure to cigarette smoke induces microRNA let-7 downregulation and cell proliferation that can be converted to cell growth arrest and apoptosis upon let-7a transfection. Therefore, microRNAs are reliable biomarkers for evaluating chemoprevention efficacy and may be used to counteract carcinogenesis.

Investigation of nuclear nano-morphology marker as a biomarker for cancer risk assessment using a mouse model

The development of accurate and clinically applicable tools to assess cancer risk is essential to define candidates to undergo screening for early-stage cancers at a curable stage or provide a novel method to monitor chemoprevention treatments. With the use of our recently developed optical technology--spatial-domain low-coherence quantitative phase microscopy (SL-QPM), we have derived a novel optical biomarker characterized by structure-derived optical path length (OPL) properties from the cell nucleus on the standard histology and cytology specimens, which quantifies the nano-structural alterations within the cell nucleus at the nanoscale sensitivity, referred to as nano-morphology marker. The aim of this study is to evaluate the feasibility of the nuclear nano-morphology marker from histologically normal cells, extracted directly from the standard histology specimens, to detect early-stage carcinogenesis, assess cancer risk, and monitor the effect of chemopreventive treatment. We used a well-established mouse model of spontaneous carcinogenesis--Apc(Min) mice, which develop multiple intestinal adenomas (Min) due to a germline mutation in the adenomatous polyposis coli (Apc) gene. We found that the nuclear nano-morphology marker quantified by OPL detects the development of carcinogenesis from histologically normal intestinal epithelial cells, even at an early pre-adenomatous stage (six weeks). It also exhibits a good temporal correlation with the small intestine that parallels the development of carcinogenesis and cancer risk. To further assess its ability to monitor the efficacy of chemopreventive agents, we used an established chemopreventive agent, sulindac. The nuclear nano-morphology marker is reversed toward normal after a prolonged treatment. Therefore, our proof-of-concept study establishes the feasibility of the SL-QPM derived nuclear nano-morphology marker OPL as a promising, simple and clinically applicable biomarker for cancer risk assessment and evaluation of chemopreventive treatment.

Abstract 1637: Silibinin causes strong efficacy towards colon cancer stem cells by modulating IL-4 caused CD44 expression

Abstract Cancer stem cells (CSC) are now recognized as the main cause for initiation, promotion and progression of most of the epithelial cancers, including colorectal cancer (CRC). Despite this fact, the efficacy of chemopreventive agents towards CSC generation leading to cancer initiation and tumorigenesis has not yet been well-defined. The present study was aimed to provide a new direction to ‘cancer chemoprevention research’ by determining whether silibinin, a natural non-toxic chemopreventive agent, has the potential to target CSC in CRC. In this regard, we determined silibinin effect on the self renewal capacity of the CSC population of CRC cell lines. We isolated different cell populations (CD44+EpCAMhigh, CD44+EpCAMlow, and CD44-EpCAMlow) from the CRC cell lines, SW480, HT-29 and LoVo, and subjected them to sphere cluster formation assays. A limiting dilution approach (cell numbers ranging from 1x104 to 300 cells) was employed to determine the isolated fraction which was enriched in CSC population. Comparative analysis of different populations, in terms of sphere formation, indicated that the CSC enriched fraction was CD44+EpCAMhigh population. Two dilutions (1000 and 3000 cells/well) of the CSC enriched CD44+EpCAMhigh, CD44+EpCAMlow and unsorted populations were then subjected to sphere cluster formation assays, in the absence or presence (a one time treatment) of silibinin (50-100µM). The % of floating spheroids (colonospheres) generated in the sphere cluster assays after 1-2 weeks were determined. The results indicated a dose dependant effect of silibinin on both number and size (volume) of colonospheres, with 100µM silibinin showing a significant reduction in the number and size of generated spheres in all three CRC cell lines. Since formation of spheroids under specific in vitro conditions is a measure of stemness, it is evident that silibinin has the potential to target the self-renewal of CSCs in CRC cell lines. Since, the role of cytokine interleukin (IL)-4 is also implicated in increased resistance of CSC to chemotherapy agents and targeting IL-4 signaling presents a rational approach for the prevention and treatment of CRC, we next determined whether silibinin effect on CSC is mediated via blocking IL-4 signaling in CRC cell lines. In this regard, treatment of all three CRC cell lines with 100µM silibinin was able to significantly reduce IL-4-induced expression of both total CD44 and its variant CD44-v6 (highly expressed in CRC tumors) in a time (12-48h) dependant manner. Subsequent in vitro studies showed this effect to be mediated in part by the inhibitory effect of silibinin on IL-4-induced activation of the transcription factor STAT-3 in terms of its Tyr705 phosphorylation in CRC cells. Together, these results suggest that more studies are needed to establish translational significance of silibinin efficacy on CRC CSCs as this agent is already in clinical-trial in CRC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1637. doi:1538-7445.AM2012-1637

Abstract 3275: Development of a new ex-vivo orthotopic model-Human breast cancer cells in mouse mammary gland organ culture

Abstract Mouse mammary organ culture (MMOC) has been classically employed for evaluating the efficacy of chemopreventive agents against development of carcinogen-induced preneoplastic lesions. Efficacy of chemopreventive agents observed in MMOC correlates well with that observed in in-vivo carcinogenesis models. In the present study, we developed a new ex-vivo Human in Mouse organ culture model which mimics in-vivo orthotopic breast cancer model. Since we introduced human breast cancer cells in mouse mammary gland, this model is termed as human Breast cancer (BCA) in Mouse Mammary Organ Culture (BCa-in-MMOC)). Three to four week old female BALB/c mice were sensitized with estradiol (1μg) + progesterone (1mg) for 9 days. On the 10th day animals were sacrificed and 2.5x104 T47Dparental or T47D aromatase overexpressing cells were injected into the fourth pair of thoracic mammary glands. The glands were excised then cultured at 37°C under 95% O2 / 5% CO2 in hMEM medium containing 10% charcoal stripped FBS/supplemented with Testosterone (1nM) and progesterone (1uM) and growth promoting hormones (5 µg insulin, 5 μg prolactin per ml medium). At the end of the experiment, the glands were fixed in formalin. The paraffin embedded sections (longitudinal) of entire glands were processed for histopathological examination (H and E stain) and immnohistochemical staining of various proteins. Mammary glands were evaluated for the presence of T47D cells, their growth pattern and their molecular responsiveness to estradiol. T47D cells (both types) injected into mammary glands were easily identified against mouse cells by intense human specific Ck-18 immunofluorescence staining. Histopathological observation of mammary gland sections showed that growth pattern of injected cancer cells was identical to that observed of breast cancer cells injected in vivo in athymic mice. Interestingly, clusters of cancer cells in the mammary gland stroma appear similar to those observed in breast tumors in women. Cancer cells injected into glands survived and continued to grow (as evident from Ki-67 immunostaining) after 15 days in culture. Cancer cells maintained their original characteristics (ER+, PR+, EGFR+, and aromatase). T47D cells with enhanced aromatase expression growing in the MMOC could metabolize testosterone to estrogen, which resulted in enhanced cell proliferation and induction of estrogen target genes such as ER and PR-B. Mouse mammary glands with T47D aromatase overexpressing cells also showed changes typical to estrogenic milieu. In summary this model provides a novel, inexpensive ex-vivo model, which could be used to study effects of therapeutic agents on the cancer cells growing in orthotopic micromilieu. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3275. doi:1538-7445.AM2012-3275

Transplacental Antioxidants Inhibit Lung Tumors in Mice Exposed to Cigarette Smoke After Birth: A Novel Preventative Strategy?

Birth is characterized by an intense oxidative stress resulting in nucleotide alterations and gene overexpression in mouse lung. We showed that cigarette smoke (CS) is carcinogenic when exposure starts soon after birth and applied this bioassay to evaluate the efficacy of chemopreventive agents. The present study evaluated whether administration of the antioxidants N-acetyl-L-cysteine (NAC) and vitamin C or ascorbic acid (AsA) during pregnancy can protect strain H Swiss mice exposed to CS after birth. Exposure to CS, for 4 months, of newborns from untreated mice resulted in significant alterations at 8 months of life, including alveolar epithelial hyperplasia, emphysema, blood vessel proliferation, microadenomas, adenomas, and malignant tumors in lung, liver parenchymal degeneration and urinary bladder epithelium hyperplasia. Treatment throughout pregnancy with either NAC, a scavenger of reactive oxygen species, or AsA, an electron donor, did not affect fertility, parity, and body weight of newborns. Prenatal antioxidants significantly inhibited most lesions in adult mice exposed to CS since birth. For instance, the incidence of emphysema was reduced from 27.5% in CS-exposed mice that were untreated during pregnancy to 7.1% and 14.0% in those treated prenatally with NAC and AsA, respectively. Lung adenomas were reduced from 34.8% to 16.7% and 9.3%, respectively. Malignant lung tumors were reduced from 13.0% to 4.7% by prenatal AsA. Liver parenchymal degeneration was reduced from 58.0% to 14.3% by prenatal NAC. These data mechanistically support a "transplacental chemoprevention" strategy, aimed at protecting the newborn from oxidative stress and the adult from CS-related diseases appearing later in life.