Rat Model Of Breast Cancer Research Articles

Cetuximab functionalized chitosan/hyaluronic acid-based nanoparticles loaded with cabazitaxel enhances anti-tumor efficacy in DMBA-induced breast cancer model in rats through spatial targeting

The article discusses the ionic gelation of cationic chitosan (CS) with anionic hyaluronic acid (HA) sodium salt to form nanoparticles in the range of 125 nm. The particles were further functionalized with cetuximab to endow it with the ability to spatially target the tumor over-expressing EGFR. Solid-state characterization of the particles using XRD, FTIR, and DSC revealed the formation of stable nanoparticles with Cabazitaxel loaded in the amorphous nanostructure. XPS study used to assess the surface characteristics indicated that the cetuximab was successfully anchored on the surface of the particle. The prepared CS-HA-Cmab-NP elicited a pH-responsive drug release behavior due to the presence of CS in the matrix. In vitro performance of the nanoparticles was evaluated on MDA-MB-231 breast cancer cell lines showed overall increase in efficacy. In vivo pharmacokinetic and anti-tumor effect evaluated in female Sprague Dawley rats indicated that the Cmab-conjugated nanoparticles improved half-life of cabazitaxel and tumor reduction capability with higher survival rate and lower reduction in body weight. The results indicate that CS/HA nanoparticles anchored with cetuximab show enhanced efficacy in reducing the breast cancer tumor in DMBA-induced breast tumor model through spatial targeting, consequently reducing the systemic toxicity.

Evaluation of tumor-suppressive properties and apoptotic functions of Mad Honey and Vincristine applications in a rat model of breast cancer

In this study, the suppressive effects of vincristine and Turkish mad honey alone and in co-applications were biochemically, hematologically, and histopathologically investigated in a mammary tumor model induced with 7,12-dimethylbenz[a]anthracene (DMBA) in rats. A total of 72 rats, 43-49 days old, were divided into 6 groups of 12 rats each. The control group (CG) consisted of healthy rats. The vehicle group (VG) received only vehicle substance and the cancer control group (CCG) was given only DMBA. DMBA and the honey group (HG) given group. DMBA and the vincristine (VinG) given group, and DMBA, the vincristine-honey group (VHG) received both Turkish mad honey and vincristine. Turkish mad honey and/or vincristine was given in the last 4 weeks of the 13-week trial period. White blood cell and lymphocyte counts differed significantly in the CCG and VG groups. Alanine transaminase and total protein levels were higher in the CCG and VinG groups. Aspartate transaminase was higher in the CCG, HG and VG groups. Caspase-3 and Bax protein levels were in the HG and VHG groups significantly higher than CCG. In caspase-8 protein level VHG significantly higher than other groups. Caspase -9 protein level was in CG and VG groups significantly lower than other groups. Bcl-xL increased more in the CCG group. Anaplasia was reduced in the HG and VinG groups, although apoptosis and other cellular damages increased. It was concluded that mad honey and vincristine could be considered together as effective therapeutic agents in this model of DMBA-induced breast cancer.

Chemopreventive effects of atractylenolide-III on mammary tumorigenesis via activation of the Nrf2/ARE pathway through autophagic degradation of Keap1

The incidence of breast cancer is increasing annually, making it a major health threat for women. Chemoprevention using natural, dietary, or synthetic products has emerged as a promising approach to address this growing burden. Atractylenolide-III (AT-III), a sesquiterpenoid present in various medicinal herbs, has demonstrated potential therapeutic effects against several diseases, including tumors, nonalcoholic fatty liver disease, and cerebral ischemic injury. However, its impact on breast cancer chemoprevention remains unexplored. In this study, we used an N-methyl-N-nitrosourea (NMU)-induced rat breast cancer model and 17β-estradiol (E2)-treated MCF-10A cells to evaluate the chemopreventive potential of AT-III on mammary tumorigenesis. AT-III inhibited mammary tumor progression, evidenced by reduced tumor volume and multiplicity, prolonged tumor latency, and the reversal of NMU-induced weight loss. Furthermore, AT-III suppressed NMU-induced inflammation and oxidative stress through the Nrf2/ARE pathway in breast cancer tissues. In vitro, AT-III effectively suppressed E2-induced anchorage-independent growth and cell migration in MCF-10A cells. Nrf2 knockdown attenuated the protective effects of AT-III, highlighting the pivotal role of Nrf2 in AT-III-mediated suppression of tumorigenesis. The mechanism involves the induction of Nrf2 expression by AT-III through the autophagic degradation of Kelch-like ECH-associated protein 1 (Keap1). Overall, the results of this study indicate that AT-III is a promising candidate for breast cancer chemoprevention and provide valuable insights into its molecular interactions and signaling pathways.

Exploration of tumor growth regression of quinoa and chia oil nanocapsules via the control of PIK3CA and MYC expression, anti-inflammation and cell proliferation inhibition, and their hepatorenal safety in rat breast cancer model

BackgroundThe second most common cancer in the world is breast cancer. Chemotherapy is used to treat breast cancer, but instances of multidrug resistance, targets that are not selective, and physicochemical issues raise doubts about its efficacy. So, the exploration of chemopreventive agents from efficient natural sources has been required. The chia and quinoa seeds have health-promoting activities that include cardio-protective, antidiabetic, and anticancer effects. Given the paramount importance of their oils and their potential bioactivities, this work aimed to assess the repressive effect of their oil nanocapsules against mammary tumors in rats. Rat models of chemically induced mammary tumors were gavaged with chia and quinoa nanocapsules for one month. The repressive effect of nanocapsules was studied by quantifying TNF-α, assessing the gene expression of proto-oncogenes (PIK3CA and MYC) using qRT-PCR, and analyzing the cell cycle in mammary tissue.ResultsThe studies clarified that the inhibition of tumors in response to quinoa and chia nanocapsules was associated with a reduction in TNF-α levels, proliferation capability, and motivation for apoptosis. Furthermore, quinoa and chia nanocapsule management repressed the activation of the MYC and PIK3CA genes. As well as nanocapsules modulated the liver enzymes and kidney function alterations induced in mammary tumor animals. Meanwhile, both oils' nanocapsules do not have an impact on the liver and kidneys of healthy rats.ConclusionsThe findings indicate that quinoa and chia nanocapsules are safe and can reduce tumor growth, suggesting a potential natural therapeutic target for breast cancer treatment.

The Herbal Combination Shu Gan Jie Yu Regulates the SNCG/ER-a/AKT-ERK Pathway in DMBA-Induced Breast Cancer and Breast Cancer Cell Lines Based on RNA-Seq and IPA Analysis.

Soothing the liver (called Shu Gan Jie Yu in Chinese, SGJY) is a significant therapeutic method for breast cancer in TCM. In this study, 3 liver-soothing herbs, including Cyperus rotundus L., Citrus medica L. var. sarcodactylis Swingle and Rosa rugosa Thunb. were selected and combined to form a SGJY herbal combinatory. To investigate the inhibiting effect of SGJY on breast cancer in vivo and vitro, and to explore the potential mechanisms. SGJY herbal combination was extracted using water. A breast cancer rat model was developed by chemical DMBA by gavage, then treated with SGJY for 11 weeks. The tumor tissue was preserved for RNA sequencing and analyzed by IPA software. The inhibition effects of SGJY on MCF-7 and T47D breast cancer cells were investigated by SRB assay and cell apoptosis analysis, and the protein expression levels of SNCG, ER-α, p-AKT and p-ERK were measured by western blotting. SGJY significantly reduced the tumor weight and volume, and the level of estradiol in serum. The results of IPA analysis reveal SGJY upregulated 7 canonical pathways and downregulated 16 canonical pathways. Estrogen receptor signaling was the key canonical pathway with 9 genes downregulated. The results of upstream regulator analysis reveal beta-estradiol was the central target; the upstream regulator network scheme showed that 86 genes could affect the expression of the beta-estradiol, including SNCG, CCL21 and MB. Additionally, SGJY was verified to significantly alter the expression of SNCG mRNA, CCL21 mRNA and MB mRNA which was consistent with the data of RNA-Seq. The inhibition effects of SGJY exhibited a dose-dependent response. The apoptosis rates of MCF7 and T47D cells were upregulated. The protein expression of SNCG, ER-α, p-AKT and p-ERK were all significantly decreased by SGJY on MCF-7 and T47D cells. The results demonstrate that SGJY may inhibit the growth of breast cancer. The mechanism might involve downregulating the level of serum estradiol, and suppressing the protein expression in the SNCG/ER-α/AKT-ERK pathway.

Hypericum alpestre extract exhibits in vitro and in vivo anticancer properties by regulating the cellular antioxidant system and metabolic pathway of L-arginine.

Conventional treatment methods are not effective enough to fight the rapid increase in cancer cases. The interest is increasing in the investigation of herbal sources for the development of new anticancer therapeutics. This study aims to investigate the antitumor capacity of Hypericum alpestre (H. alpestre) extract in vitro and in vivo, either alone or in combination with the inhibitors of the l-arginine/polyamine/nitric oxide (NO) pathway, and to characterize its active phytochemicals using advanced chromatographic techniques. Our previous reports suggest beneficial effects of the arginase inhibitor NG-hydroxy-nor- l-arginine and NO inhibitor NG-nitro-Larginine methyl ester in the treatment of breast cancer via downregulation of polyamine and NO synthesis. Here, the antitumor properties of H. alpestre and its combinations were explored in vivo, in a rat model of mammary gland carcinogenesis induced by subcutaneous injection of 7,12-dimethylbenz[a]anthracene. The study revealed strong antiradical activity of H. alpestre aerial part extract in chemical (DPPH/ABTS) tests. In the in vitro antioxidant activity test, the H. alpestre extract demonstrated pro-oxidant characteristics in human colorectal (HT29) cells, which were contingent upon the hemostatic condition of the cells. The H. alpestre extract expressed a cytotoxic effect on HT29 and breast cancer (MCF-7) cells measured by the MTT test. According to comet assay results, H. alpestre extract did not exhibit genotoxic activity nor possessed antigenotoxic properties in HT29 cells. Overall, 233 substances have been identified and annotated in H. alpestre extract using the LC-Q-Orbitrap HRMS system. In vivo experiments using rat breast cancer models revealed that the H. alpestre extract activated the antioxidant enzymes in the liver, brain, and tumors. H. alpestre combined with chemotherapeutic agents attenuated cancer-like histological alterations and showed significant reductions in tumor blood vessel area. Thus, either alone or in combination with Nω -OH-nor- l-arginine and Nω -nitro- l-arginine methyl ester, H. alpestre extract exhibits pro- and antioxidant, antiangiogenic, and cytotoxic effects.

Incorporation of exemestane into ternary nanosponge system for enhanced anti-tumor potential in breast cancer

The present investigation reports the potential of exemestane loaded cyclodextrin based nanosponges for the treatment of breast cancer. Fourier transform infrared, and nuclear magnetic resonance (NMR) spectroscopic analysis confirmed the encapsulation of ring B, C, and D of exemestane in the nanosponge cavity. In vitro studies demonstrated a 6.58-folds increase in the aqueous solubility and a 1.76-folds increase in the dissolution of exemestane in the optimized nanosponge formulation EF2. It also exhibited enhanced cytotoxicity in MCF-7 cell line. Pharmacokinetic studies revealed a 1.37-fold increase in C max and a 2.10-fold increase in oral bioavailability of EF2, as compared to its marketed product Aromasin®. Concomitantly, this nano-formulation reduced the tumor burden to 45.71% in a DMBA-induced breast cancer rat model. This EF2-treatment also improved the hematological parameters of the animals. Histopathology of breast tissue also presented reduction in characteristic cytoarchitectural features of breast tumor. In vivo toxicity studies demonstrated reduced hepatotoxicity of the nanosponge formulation when compared with Aromasin®. These results were further supported by histological studies of excised liver tissues, where the size of hepatocytes in EF2-treated animals was like the normal hepatocyte size. In conclusion, the encapsulation of exemestane in β-cyclodextrin nanosponge along-with HPMC E5 improved its aqueous solubility, bioavailability, and ultimately therapeutic efficacy for the treatment of breast cancer.

Mesoporous silica nanoparticles boost aggressive cancer response to hydrophilic chlorin e6-mediated photodynamic therapy

BackgroundChlorin e6 trisodium salt (Ce6) is a newly developed hydrophilic photosensitizer designed to mediate anticancer photodynamic therapy (PDT). The response of different cancer types and strategies to boost anticancer efficiency of Ce6-PDT are poorly studied.ObjectivesThis study aimed to investigate the response of different cancer types to Ce6-PDT, identify the unresponsive ones, and develop a nanosystem for response enhancement.MethodsSk-Br-3, MCF-7, U87, and HF-5 cells were tested in 2D cell cultures. Ce6 uptake, PDT-mediated phototoxicity, ROS production, caspase 3/7 levels, and cell death mode were examined. Furthermore, U87 spheroids were treated with Ce6-PDT. Mesoporous silica nanoparticles (MSN) were synthesized and loaded with Ce6. Cellular uptake and phototoxicity of MSN-Ce6 were compared to free Ce6 in vitro and in vivo.ResultsCe6 was detectable in the cell cytoplasm within 15 min. U87 cells showed the highest Ce6 cellular uptake. Upon Ce6-PDT, U87 cells were the most responsive ones with an 11-fold increase in ROS production. Here, 5 µM Ce6 and 4 J/cm2 were enough to reach IC50. Ce6-PDT induced both necrotic and caspase-dependent apoptotic cell death and 75% reduction of spheroids volume. Also, MCF-7 and HF-5 cells responded well to Ce6-PDT treatment. Sk-Br-3 breast cancer cells, on the other hand, were the least responsive ones with 80% viability after treatment (5 µM Ce6, 8 J/cm2). However, MSN-Ce6 conjugates increased Sk-Br-3 cellular uptake of Ce6 sevenfold decreasing the IC50 irradiation dose by an order of magnitude. In a very aggressive breast cancer rat model, MSN-Ce6-PDT treatment led to suppression of tumor volume by 50% and elevation of both Bax and caspase 3 by 90% compared to the control while the corresponding values for Ce6-PDT were 30% and 70%, respectively.ConclusionThe newly developed hydrophilic chlorin and even more its MSN conjugate show high activities in anticancer PDT.

Anti-cancer effect of Rumex obtusifolius in combination with arginase/nitric oxide synthase inhibitors via downregulation of oxidative stress, inflammation, and polyamine synthesis.

Cancer continues to be a leading cause of death worldwide, making the development of new treatment methods crucial in the fight against it. With cancer incidence rates increasing worldwide, ongoing research must focus on identifying new and effective ways to prevent and treat the disease. The combination of herbal extracts with chemotherapeutic agents has gained much interest as a novel strategy to combat cancer. Rumex obtusifolius L. is a wild plant known for its medicinal properties and is widely distributed worldwide. Our preclinical evaluations suggested that R. obtusifolius seed extracts possessed cancer-inhibiting properties and we also evaluated the beneficial effects of the arginase inhibitor NG-hydroxy-nor-L-arginine and nitric oxide inhibitor NG-nitro-L-arginine methyl ester in the treatment of breast cancer. The current study aimed to combine these observations and evaluate the antioxidant and antitumor properties of R. obtusifolius extracts alone and in combination with the arginase and nitric oxide synthase inhibitors. Metabolic characterization of the plant extract using a liquid chromatography/high-resolution mass spectrometry advanced system revealed the presence of 240 phenolic compounds many of which possess anticancer properties, according to the literature. In vitro studies revealed a significant cytotoxic effect of the R. obtusifolius extracts on the human colon (HT29) and breast cancer (MCF-7) cell lines. Thus, a new treatment approach of combining R. obtusifolius bioactive phytochemicals with the arginase and nitric oxide synthase inhibitors NG-nitro-L-arginine methyl ester and/or NG-hydroxy-nor-L-arginine, respectively, was proposed and could potentially be an effective way to treat breast cancer. Indeed, these combinations showed immunostimulatory, antiproliferative, antioxidant, anti-inflammatory, and antiangiogenic properties in a rat breast cancer model.

Abstract P3-08-08: Establishment of a Breast Cancer Rat Model of Chemotherapy-Induced Cardiotoxicity

Abstract Establishment of a Breast Cancer Rat Model of Chemotherapy-Induced Cardiotoxicity Katherine Wallis, Marjan Boerma, Antiño Allen, Joseph Su, Sam Makhoul, Ping-Ching Hsu Background. Doxorubicin (DOX) is a highly effective chemotherapy agent that is commonly used in combination with precision medicine to treat a wide range of cancers, including 32% of breast cancer (BC) cases. Although the treatment has greatly increased the number of long-term cancer survivors, it has also increased the number of patients experiencing DOX-induced cardiotoxicity (DIC). Animal models have played a vital role in the development of basic and translational breast cancer research in humans, and rat is a preferred animal to study breast cancer for the similarity to human neoplasms and metabolism. However, most studies were established using mouse models, male rats and commonly non-cancer bearing animals. Currently no single universal cancer-bearing female rat model is utilized for DOX-induced chronic cardiotoxicity. The number of cancer cells to be injected to mimic human experience is not known. Large numbers of injected cancer cells grow quickly and kill the animal before the investigators have the time to assess for cardiac toxicity. Here we aimed to identify the minimal number of cancer cell injections to mimic tumor growth of BC patients and to achieve chemotherapy-induced toxicities. Methods. Various numbers (5 × 104 – 1 × 106) of MAT B III adenocarcinoma cancer cells derived from Fischer 344 (F344) rats were injected in the mammary fat pad of female F344 rats, followed by six intraperitoneal (IP) injections of either DOX (1.25mg/kg per dose) or saline post tumor cell injection. The treatments were administered on day 4 from cell injection, and every third day after for a total of six injections. Echocardiography (Vevo 3100, VisualSonics) was conducted before and after the end of chemotherapy. Results. Tumor burden reduced significantly among rats treated with DOX compared with the saline group. In the Kaplan-Meier survival curve, an injection of 5 × 104 cancer cells was associated with tumor growth but the tumor burden was low enough to allow long-term follow-up of both DOX and saline treated rats and assess signs of cardiotoxicity. At 4 days after the final DOX administration, increased left ventricular ejection fraction and fractional shortening in the absence of increased stroke volume were an indication of adverse cardiac effects in the DOX animals but not the saline-treated rats. Plasma was collected from the animals to be subjected to metabolomics testing so that a preliminary assessment of metabolites associated with DIC could be gathered. Conclusion. We have established a breast cancer rat model in which DIC can be studied. Research in this model can aid in identifying mechanisms of DIC and testing novel interventions. Citation Format: Katherine Wallis, Marjan Boerma, Antino Allen, Joseph Su, Sam Makhoul, Ping-Ching Hsu. Establishment of a Breast Cancer Rat Model of Chemotherapy-Induced Cardiotoxicity [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-08.

Metastasis from the tumor interior and necrotic core formation are regulated by breast cancer-derived angiopoietin-like 7

Necrosis in the tumor interior is a common feature of aggressive cancers that is associated with poor clinical prognosis and the development of metastasis. How the necrotic core promotes metastasis remains unclear. Here, we report that emergence of necrosis inside the tumor is correlated temporally with increased tumor dissemination in a rat breast cancer model and in human breast cancer patients. By performing spatially focused transcriptional profiling, we identified angiopoietin-like 7 (Angptl7) as a tumor-specific factor localized to the perinecrotic zone. Functional studies showed that Angptl7 loss normalizes central necrosis, perinecrotic dilated vessels, metastasis, and reduces circulating tumor cell counts to nearly zero. Mechanistically, Angptl7 promotes vascular permeability and supports vascular remodeling in the perinecrotic zone. Taken together, these findings show that breast tumors actively produce factors controlling central necrosis formation and metastatic dissemination from the tumor core.

Modifiers of radiation effects on breast cancer incidence revealed by a reanalysis of archival data of rat experiments.

Cancer risk after exposure to ionizing radiation can vary between individuals and populations, but the impact of factors governing those variations is not well understood. We previously conducted a series of carcinogenesis experiments using a rat model of breast cancer, in which 1654 rats born in 2002-2012 were exposed to γ rays at various doses and ages with or without non-radiation factors including high-fat diet, parity and chemical carcinogens. We herein reanalyze the incidence data from these archival experiments to clarify the effect of age at exposure, attained age, radiation dose and non-radiation factors (i.e. fat, parity, chemicals and birth cohorts) on radiation-related mammary cancer incidence. The analysis used excess relative risk (ERR) and excess absolute risk (EAR) models as well as generalized interaction models. Age-at-exposure dependence displayed a peak of susceptibility at puberty in both the ERR and EAR models. Attained age decreased ERR and increased EAR per unit radiation dose. The dose response was concordant with a linear model. Dietary fat exhibited a supra-multiplicative interaction, chemicals represented a multiplicative interaction, and parity and birth cohorts displayed interactions that did not significantly depart from additivity or multiplicativity. Treated as one entity, the four non-radiation factors gave a multiplicative interaction, but separation of the four factors significantly improved the fit of the model. Thus, the present study supports age and dose dependence observed in epidemiology, indicates heterogenous interactions between radiation and various non-radiation factors, and suggests the potential use of more flexible interaction modeling in radiological protection.

Amelioration of Doxorubicin-Induced Cognitive Impairment by Quercetin in a Rat Model of Breast Cancer

The management of breast cancer by chemotherapeutic agents has significantly increased the survival rates and, at the same time, raised concerns about the side effects caused by these agents on healthy tissues. Chemotherapy-induced cognitive impairment resulting from non-CNS malignancies like breast cancer has emerged as a significant challenge among cancer survivors due to its negative impact on the quality of day-to-day life activities. Using doxorubicin-based chemotherapy as a preclinical model for chemotherapy-induced cognitive impairment, we assessed the effect of quercetin on behavioral memory alterations in tumor-bearing female rats in vivo and changes in neurite length and apoptosis in PC12 cell lines in vitro. Quercetin is purported to have neuroprotective effects in different preclinical models of human neurological conditions because of its possible anti-inflammatory and antioxidant properties. Mammary carcinoma was induced by intraperitoneal administration of N-methyl-N-nitrosourea followed by doxorubicin administration once in 5 days (50 days). Pre-treatment with quercetin began 1 week before the chemotherapy and continued till the end of the chemotherapy cycle. Mechanistically, quercetin produced protection against doxorubicin-induced neurotoxicity by decreasing apoptosis and had a neurogenic effect evidenced by the prevention of toxicant-induced inhibition of neurite establishment. Quercetin reversed episodic and spatial memory deficits caused by doxorubicin treatment assessed by novel object recognition memory and Morris water maze, respectively. Taken together, our findings suggest that cytotoxic effects of doxorubicin may be a contributor to neurogenic impairment in tumor-bearing animals, leading to memory deficits. Therefore, quercetin could be a promising therapeutic strategy for doxorubicin-related cognitive impairment, thus imparting hope for improved quality of life among cancer survivors.Graphical

Abstract IA035: DCIS to IDC progression - a key step of immune escape

Abstract The cellular and molecular mechanisms underlying ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) transition are poorly understood. We have analyzed genetic, gene expression, epigenetic profiles of cells composing the tumor and identified cell type-specific changes during tumor progression. Importantly, we detected significant changes in the immune cell composition during tumor progression implying immune escape in invasive tumors and active adaptive immune response in DCIS. We demonstrate that these progression stage-specific alterations are also present in a rat model of breast cancer and that combined targeting of multiple components of the tumor ecosystem increases the efficacy of anti-cancer therapies. Our data highlight the role of microenvironmental, especially immune-related changes in driving breast tumor progression and emphasize the importance of studying pre-invasive tumors. Citation Format: Kornelia Polyak. DCIS to IDC progression - a key step of immune escape [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr IA035.

Transpapillary iontophoretic delivery of resveratrol loaded transfersomes for localized delivery to breast cancer.

Localized drug delivery to the breast tissues is an area of interest as a potential route to ensure site-specific drug delivery. Transpapillary delivery via the mammary papilla has advantages as most breast tumors arise from the milk ducts. The present study explored the plausibility of transpapillary delivery of a phytochemical, resveratrol (RVT), for breast cancer treatment. RVT was encapsulated within the transfersomes (RVT-TRF) to enable a sustained release of the drug using the biomaterial soya phosphatidylcholine (SPC). Iontophoresis was applied to further accelerate the penetration of the RVT-TRF across the mammary papilla to the breast tissue. The RVT-TRF development was optimized by the Design of Experiments (DoE) approach. The in vitro transpapillary iontophoresis study on porcine mammary papilla showed an enhanced penetration of RVT-TRF when compared to passive diffusion. The transpapillary delivery was further confirmed from the in vitro fluorescent microscopy study using FITC conjugated RVT-TRF. The optimized RVT-TRF delivered via transpapillary route showed a higher Cmax and AUC when compared to pure RVT given orally. A significant reduction in the tumor volume and the serum biomarker CA 15-3, when evaluated in a chemically induced breast cancer rat model, provided evidence of the effectiveness of the developed formulation when delivered locally via transpapillary route compared to the oral route. Thus the developed RVT-TRF administered via transpapillary iontophoresis technique is a promising strategy enabling a localized delivery for effective breast cancer therapy.

Anti-cancer effect of in vivo inhibition of nitric oxide synthase in a rat model of breast cancer.

Increased expression of nitric oxide synthase (NOS) is associated with different cancers such as cervical, breast, lung, brain, and spinal cord. Inhibition of NOS activity has been suggested as potential tool to prevent breast cancer. The anti-tumor therapeutic effect of L-nitro arginine methyl ester (L-NAME), NOS inhibitor, using in vivo models is currently under investigation. We hypothesized that L-NAME will show an anti-tumor effect by delaying a progression of breast cancer via a modulation of cell death and proliferation, and angiogenesis. We used a novel model of anti-cancer treatment by the administration of L-NAME (30mg/kg in a day, intraperitoneal) injected every third day for five weeks to rat model of 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast tumor. Concentrations of nitrite anions, polyamines, malondialdehyde, NH4+ levels, and arginase activity in the blood were decreased in DMBA + L-NAME-treated rats compared with DMBA rats. The mortality rates, tumor number, weight, and volume, as well as the histopathological grade of breast cancer were also significantly reduced. In addition, L-NAME treatment showed a delay in tumor formation, and in body weight compared with rats administrated only with DMBA. In conclusion, our data show that L-NAME is a promising anti-cancer agent to treat breast cancer, which can lead to development of anti-tumor therapeutic tools in future.

Targeted delivery of doxorubicin by Thermo/pH-responsive magnetic nanoparticles in a rat model of breast cancer

The novel folate conjugated Thermo/pH-responsive magnetic nanoparticles (folate-poly-MNPs) have been developed as a potential nanocarrier for improving site-specific drug delivery, tumor drug accumulation, and therapeutic effects while reducing the adverse effects of conventional drug delivery systems. To evaluate the anticancer efficacy of developed tumor-targeted drug delivery system, forty rat models of breast cancer received saline as control, DOX, DOX-poly-MNPs, and DOX-folate-poly-MNPs at a dose of 2 mg/kg/48 h. The DOX-folate-poly-MNPs showed a significant increase in protein expression of BAX and C-caspase-3 with concomitant downregulation of Bcl-2 expression and ki67 proliferation index compared to the DOX group. The synergistic antitumor efficacy of passive and active drug targeting led to enhanced drug uptake, increased tumor cell apoptosis, decreased tumor volume, and a prolonged survival rate in animals, suggesting that DOX-folate-poly-MNPs may prove to be a promising nanomedicine for the smart treatment of breast cancer in the future.

Multimodal Bone Metastasis-associated Epidermal Growth Factor Receptor Imaging in an Orthotopic Rat Model.

Purpose To develop multimodality imaging techniques for measuring epidermal growth factor receptor (EGFR) as a therapy-relevant and metastasis-associated molecular marker in triple-negative mammary adenocarcinoma metastases. Materials and Methods An orthotopic bone metastasis EGFR-positive, triple-negative breast cancer (TNBC) model in rats was used for bioluminescence imaging, SPECT/CT, PET/CT, and MRI with quantitative analysis of transcripts (n = 22 rats). Receptor-specific MRI of EGFR expression in vivo was performed by acquiring spin-echo T1-weighted images after sequential administration of a pair of anti-EGFR antigen binding fragments, F(ab')2, conjugated to either horseradish peroxidase or glucose oxidase, which have complementing activities, as well as paramagnetic (gadolinium[III]-mono-5-hydroxytryptamide of 2,2',2''-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, or Gd-5HT-DOTAGA) or positron-emitting (gallium 68-5HT-DOTAGA) substrates for MRI and PET/CT imaging, respectively. EGFR expression was confirmed by quantitative reverse transcriptase polymerase chain reaction and immunohistochemical analyses to compare with image findings. Results After surgical intraarterial delivery of TNBC cells, rats developed tumors that diverged into either rapidly growing osteolytic or slow-growing nonosteolytic tumors. Both tumor types showed receptor-specific initial MRI signal enhancement (contrast-to-noise ratio) that was three to six times higher than that of normal bone marrow (29.4 vs 4.9; P < .01). Micro PET/CT imaging of EGFR expression demonstrated a high level of heterogeneity with regional uptake of the tracer, which corresponded to region-of-interest MRI signal intensity elevation (121.1 vs 93.3; P < .001). Analysis of metastases with corroboration of imaging results showed high levels of EGFR protein and messenger RNA, or mRNA, expression in the invasive tumor. Conclusion Convergence of multimodal molecular receptor imaging enabled comprehensive assessment of EGFR overexpression in an orthotopic model of TNBC metastasis. Keywords: Animal Studies, Molecular Imaging-Cancer, MR-Contrast Agent, Radionuclide Studies, Skeletal-Appendicular, Metastases Supplemental material is available for this article. © RSNA, 2021.

Valproic acid triggers radiation-induced abscopal effect by modulating the unirradiated tumor immune microenvironment in a rat model of breast cancer.

An abscopal effect occurs when localized radiotherapy causes the regression of tumors distant from the irradiated site. However, such a clinically detectable abscopal effect from radiotherapy alone is rare. This study investigated whether valproic acid ([VPA], a histone deacetylase inhibitor [HDACi]) treatment can stimulate radiation-induced abscopal effect. We used 7,12-dimethylbenz[a]anthracene, a typical environmental carcinogen, to establish a rat model with multiple breast tumors. Only one tumor received 8 Gy fractionated doses of X-rays (2 Gy daily fractions over four days) and 200mg/kg VPA was administered intraperitoneally. We monitored the growth of both irradiated and unirradiated tumors after treatments. The unirradiated tumor was collected for hematoxylin and eosin (HE) staining, immunohistochemistry (IHC) (CD8, Granzyme B, Cleaved Caspase-3, BrdU, Ki67, F4/80 and CD68), double immunofluorescence (F4/80 and CD86), Western blot (Cleaved Caspase-3) and qRT-PCR (CD86, CD163, IL-1β, IL-6, IL-12, IL-23, IL-10, TGF-β) analysis. We found ionizing radiation (IR) + VPA treatment inhibited both irradiated and unirradiated tumor growth as compared to IR alone. Such observe abscopal effect was mediated by the recruitment of activated CD8+ T cells into the unirradiated tumor sites, which released Granzyme B to cause tumor cell apoptosis. Furthermore, IR + VPA treatment led to macrophages infiltration into the unirradiated tumor sites and polarization to M1 phenotype, resulted in increased levels of pro-inflammatory cytokines such as IL-1β and IL-12, and decreased levels of anti-inflammatory cytokines such as IL-10 and TGF-β. Our data supports the proposition that VPA may be a potential therapeutic candidate to trigger radiation-induced abscopal effect by modulating the unirradiated tumor immune microenvironment.

The Valproate Mediates Radio-Bidirectional Regulation Through RFWD3-Dependent Ubiquitination on Rad51.

Ionizing radiation (IR) can induce DNA double-strand breaks (DSBs) in tumor cells during radiotherapy (RT), but the efficiency of RT is limited because of the toxicity to normal cells. Locating an adjuvant treatment to alleviate damage in normal cells while sensitizing tumor cells to IR has attracted much attention. Here, using the 7,12-dimethylbenz[α]anthracene (DMBA)-induced malignant transformed MCF10A cells, we found that valproate (VPA), a histone deacetylase inhibitor (HDACi), radiosensitized transformed cells while alleviated IR-induced damage in normal cells at a safe dose (0.5 mM). We further demonstrated the decrease of homologous recombination (HR)-associated Rad51 in the transformed cells was related to the increase of its ubiquitination regulated by E3 ligase RFWD3 for the radiosensitization, which was opposite to normal cells, indicating that RFWD3-dependent ubiquitination on Rad51 was involved in the VPA-mediated radio-bidirectional effect. Through DMBA-transformed breast cancer rat model, VPA at 200 mg/kg radiosensitized tumor tissue cells by increasing RFWD3 and inhibited Rad51, while radioprotected normal tissue cells by decreasing RFWD3 and enhanced Rad51. In addition, we found high-level Rad51 was associated with tumorigenesis and poor prognosis in breast cancer patients. Our findings uncovered RFWD3-dependent Rad51 ubiquitination was the novel mechanism of VPA-mediated radio-bidirectional effect, VPA is a potential adjuvant treatment for tumor RT.