Chitosan Nanoparticles of Paclitaxel for the Treatment against MDA-MB-231 Breast Cancer Cells via Oral Nanomedicine: Fabrication and Preclinical Assessment

Breast cancer (BCa) and prostate cancer (PCa) are both hormone driven cancers with similar etiology and tumor microenvironment inflammation promotes BCa and PCa progression. Interleukin-1 (IL-1) is an inflammatory cytokine present in the tumor microenvironment and IL-1 is elevated in BCa and PCa patient tumor and/or blood serum and correlates with poor prognosis. We have shown that IL-1 represses the BCa and PCa therapeutic targets, Estrogen Receptor Alpha (ERa) and Androgen Receptor (AR), respectively, in PCa and BCa cell lines; yet the cells remain viable. Thus, IL-1 may promote treatment resistance and disease progression. The pro-survival protein Sequestome-1 (SQSMT1/p62) is also upregulated in both BCa and PCa cell lines exposed to IL-1, suggesting that BCa and PCa cells have evolved a shared response to IL-1 and hormone receptor loss. RNA sequencing of an IL-1-treated PCa cell line revealed an IL-1-modulated gene suite predicted to confer AR-independent tumorigenicity. We performed RT-qPCR in PCa cell lines for several select genes to confirm the RNA sequencing results and given the similar etiology and IL-1 regulation of ERa, AR, and p62 expression in BCa and PCa cell lines, we presumed that our select genes would be similarly regulated by IL-1 in BCa cells. However, outside of ERa, AR, and p62 expression, our select genes did not show similar IL-1 regulation in BCa cell lines, suggesting that IL-1 regulates a unique set of genes that could contribute to ERa-independent tumorigenicity in BCa cells. Therefore, to identify a BCa-specific IL-1-modulated gene suite, we performed RNA sequencing on an IL-1-treated ERα+ BCa cell line and compared gene expression changes with 1) basal gene expression in an ERa- BCa cell line and 2) our IL-1-modulated PCa-specific gene suite. Our bioinformatics analysis revealed pathways that are predicted to promote ERa-independent tumorigenicity in BCa cells and investigations are underway to demonstrate the functional significance of our gene expression data. Taken together, our studies provide insight into the mechanistic function of IL-1 in PCa and BCa resistance to hormone receptor-targeted therapies and tumor progression. Citation Format: Afshan Fathima Nawas, Mohammed Kanchwala, Shayna Thomas-Jardin, Vanessa Anunobi, Ally Wong, Chao Xing, Nikki Delk. Identification of Interleukin-1 (IL-1) induced gene expression pattern in breast cancer (BCa) cells [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 3444.

Box Behnken optimization of cubosomes for enhancing the anticancer activity of metformin: Design, characterization, and in-vitro cell proliferation assay on MDA-MB-231 breast and LOVO colon cancer cell lines

Adenoviral vectors with E1A regulated by tumor-specific promoters are selectively cytolytic for breast cancer and melanoma.

BACKGROUND: CDK4/6 regulates the G1-S phase transition by phosphorylating the retinoblastoma protein (Rb). Given their potent clinical efficacy, CDK4/6 inhibitors used in combination with hormone receptor (HR) blockade (with an aromatase inhibitor or fulvestrant) are emerging as the standard of care for patients with metastatic HR-positive breast cancers. The CDK4/6 inhibitors palbociclib and ribociclib are FDA-approved for use in HR-positive breast cancer patients, and abemaciclib is currently in phase III trials. We observed that approximately 74% (25/34) of breast cancer cell lines had high phosphorylated Rb (phospho-Rb) expression levels and that triple-negative breast cancer (TNBC) cell lines often expressed phospho-Rb, suggesting that targeting phospho-Rb via CDK4/6 inhibition may be effective against TNBC. The histone deacetylase (HDAC) inhibitors increase p21Cip1 levels, promoting proteasomal degradation of cyclin B1 and resulting in G2/M arrest. Entinostat is an oral, class 1, selective HDAC inhibitor currently in phase III testing in HR-positive breast cancer. Preclinical and clinical data demonstrate that entinostat, in combination with HR blockade, has anticancer activity. Our group recently reported that entinostat combined with other anticancer drugs induced apoptosis via induction of proapoptotic proteins such as Noxa and Bim in breast cancer cell lines. Based on these findings, we hypothesized that entinostat-induced apoptosis and palbociclib-induced cell cycle arrest synergize to produce enhanced antitumor effects in estrogen receptor (ER)-positive breast cancer and TNBC cell lines with high phospho-Rb expression levels. METHODS: We assessed the combination antitumor effects and their mechanisms via CellTiter Blue and sulforhodamine B assays, flow cytometry, apoptosis (caspase 3/7) assays, anchorage-independent growth assays, Western blotting, reverse phase protein array (RPPA), and mammary fat pad xenograft mouse models. RESULTS: RPPA data showed that ER-positive and TNBC cell lines more often expressed phospho-Rb than did other breast cancer cell subtypes (7/10 and 8/17 cell lines, respectively). We found that the combination of entinostat and palbociclib synergistically inhibited tumor cell proliferation (combinational index less than 1.0), reduced in vitro colony formation (P < 0.05), inhibited in vivo tumor growth in ER-positive MCF-7 breast cancer cells (P < 0.05), and inhibited tumor growth in TNBC xenograft mouse models (MDA-MB-231) more effectively than did either drug alone. CONCLUSION: Taken together, our data provide evidence that combining entinostat with palbociclib enhances the antitumor effects of these drugs. Along with our continued effort to determine predictive biomarkers, our findings justify conducting a clinical trial of combination treatment with entinostat and palbociclib in patients with ER-positive breast cancer or TNBC. Citation Format: Lee J, Lim B, Pearson T, Tripathy D, Ordentlich P, Ueno NT. The synergistic antitumor activity of entinostat (MS-275) in combination with palbociclib (PD 0332991) in estrogen receptor-positive and triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-21-15.

The pro-proliferative effect of interferon-γ in breast cancer cell lines is dependent on stimulation of ASCT2-mediated glutamine cellular uptake

Objective: The objective of this study was to prepare ceftriaxone sodium chitosan nanoparticles (CS-NP) from different drug and polymer ratios and analyze their physicochemical characteristics.Methods: Ceftriaxone sodium loaded chitosan nanoparticles were prepared using chitosan as a polymer and tri sodium polyphosphate (TPP) as cross linking agent by ionic cross linking and coacervation with the aid of sonication. Various trials have been carried out for the confirmation of nanoformulation. Parameters such as the zeta potential, polydispersity, particle size, entrapment efficiency, in vitro drug release Thermo gravimetric analysis and scanning electron microscope of the nanoparticles were assessed for confirmation of nanoformulation.Results: The formulated nanoparticles showed mean particle size, polydispersity index and zeta potential to be 183.1±8.42 nm, 0.212±0.05, +38.5±1.6 mV respectively and the drug loading was found to be 46.42±10 %. In vitro drug release was showed a biphasic release pattern with initial burst release followed by sustained release of formulated nanoparticles. The cumulative percentage of drug release was about 83.08 %.Conclusion: Formulation F2 was found to be the best formulation with a higher cumulative percentage of drug release. Modified ionic gelation method can be utilized for the development of chitosan nanoparticles of ceftriaxone sodium. Polymer and crosslinking agent concentrations and sonication time are rate-limiting factors for the development of the optimized formulation. The chitosan nanoparticles developed would be capable of sustained delivery of ceftriaxone sodium.

This article presents a new and environmentally friendly method for generating DH-CdSNPs (cadmium sulfide nanoparticles) ranging from 5-10 nm in size. A green synthesis method for the development of inorganic nanoparticles was developed a few years back for their applications in diverse fields, such as medicine, bioimaging and remediation. The biogenic synthesis of these nanoparticles containing daruharidra (Berberis aristata) and cadmium sulfide is an effective alternative. By employing Daruharidra extract as a herbal analog, we aim to minimize the risks and adverse effects that come along with the use of other chemically synthesized nanoparticles. This study's main goal was to investigate the potential of these nanoparticles as powerful antibacterial and anticancer agents. We used a crude powdered daruharidra extract as a stabilizer ingredient to create CdSbased nanoformulations in an environmentally responsible way. By exposing the breast cancer cell line (MDAMB-231) and ovarian teratocarcinoma cell line (PA1) to these nanoformulations, we were able to evaluate their anticancer activities. Additionally, flow cytometry analysis was conducted to scrutinize the process of cell cycle arrest and apoptosis in reference to anticancer studies. Furthermore, DH-CdSNPs were applied on different gram-positive as well as gramnegative bacteria in a disc diffusion assay to ascertain their antibacterial activity. Nanoparticles were tested on bacterial strains to check if they were resistant after the MIC or minimum inhibitory concentration. The cytotoxicity of nanoparticles was tested by MTT assay. The impact of increasing concentrations of NPs on cell lines was tested, revealing a cytotoxic effect. The half-maximal inhibitory concentration values for a 24-hour treatment were determined to be 95.74μg/ml for ovarian cancer cells and 796.25 μg/ml for breast cancer cells. Treatment with DH-CdSNP resulted in a noteworthy increase in early apoptotic cells, with percentages rising from approximately 3% to 14.5% in ovarian cancer cell lines and from 4% to 13.6% in breast cancer cell lines. Furthermore, the NPs induced arrest of the cell cycle, specifically in the interphase of G2 and mitosis phase, with DNA damage observed in sub G1 in ovarian cancer cells and G0/G1 arrest observed in breast cancer cells. Additionally, the NPs exhibited exceptional potency against both gram-positive as well as gram-negative bacteria. Less research has been done on using bioinspired DH-CdSNP to deliver anticancer medications. The amalgamation of plant extract and the DH-CdSNP could cause a paradigm shift in the cancer therapy approach. The findings revealed that the biosynthesized DH-CdSNP limited the growth of human breast and ovarian cancer cells. This property can be further investigated against a variety of additional cell lines to determine whether this property makes the DH-CdSNP a promising treatment alternative. The results obtained from these nanoformulations exhibit faster efficacy compared to traditional medications.

We focused on qualitatively exploring the basic mechanisms involved in the Ionic gelation (IG) process, a method quite frequently used for synthesis of chitosan (CS) microparticles (MPs) and nanoparticles (NPs).We synthesized CS MPs and NPs using the Ionic gelation and microemulsion methods, and characterized the CS NPs and MPs at different stages of formulation using scanning electron microscopy (SEM) and fluorescence microscopy. Fourier Transform Infrared (FTIR) analysis was carried out to confirm effective cross-linking. Moreover, for the first time, we reported the mechanisms of IG technique for CS NP and MP synthesis with qualitative proof: (1) Complex formation of long chain oligomers with polyanions (long beaded structures) (2) cleavages at weak sites on addition of acid (HCl) (3) formation of CS NPs on chain scission.The versatility of IG for the synthesis of CS MPs and NPs was proved and compared with the microemulsion technique, thereby enhancing the wide spectrum of its use in therapeutics and biomedical applications.

Background We investigate the immunogenic properties of chitosan and liposome nanoparticles as adjuvant codelivery against a commercial pneumococcal conjugate vaccine (PCV) in an animal model. Methods The chitosan and liposome nanoparticles were prepared by ionic gelation and dry methods, respectively. The PCV immunization was performed intradermally in the presence of adjuvants and booster injections which were given without an adjuvant. The Quil-A® was used as a control adjuvant. The ELISA was performed to measure the antibodies against pneumococcal type 14 polysaccharide (Pn14PS). Results The level of total antibodies against Pn14PS antigen was no different between the mouse groups with or without adjuvant codelivery. Codelivery of the PCV with chitosan nanoparticles as well as the Quil-A adjuvant elicited IgG1, IgG2a, IgG2b, and IgG3 antibodies. Meanwhile, codelivery of liposome nanoparticles elicited mainly IgG1 antibodies against the Pn14PS. Conclusions The chitosan and liposome nanoparticles as adjuvant codelivery were successfully synthesized. These nanoparticles have different shapes in particle formation, liposome nanoparticle with their unilamellar shape and chitosan nanoparticles in large shape due to the aggregation of small-size particles. Codelivery of chitosan nanoparticles has more effect on the IgG subclass antibody production than that of liposome nanoparticles in a mouse model.

Background: Metastatic disease is the primary cause of breast cancer (BC) mortality. Tumor-stromal cell interactions play a pivotal in tumor initiation, progression and metastasis. Cancer-associated fibroblasts (CAFs) are the majority of stroma in BC and are critical to BC tumorigenicity and malignancy. Metastasis occurs due to the transport of circulating tumor cells (CTC) and clusters of CTCs through the vasculature. We recently identified circulating CAFs (cCAFs) as a novel circulating biomarker associated with metastatic BC. cCAFs not only circulate individually, in BC patient blood and in blood from both spontaneous and xenograft murine BC models, but cCAFs are also found in clusters with CTCs. In this study, we examine the egress of CAFs and follow them through to metastatic sites, and evaluate the ability of BC cell subtype/metastatic propensity to influence cCAF and cCAF/CTC cluster egress. Methods: We used NSG mice with orthotopic xenograft implantation of BC cells, primary CAF cell lines, or co-implantation of BC and CAF cell lines. We used two different BC cell lines, the nonmetastatic BC cell line, MCF-7, and the highly metastatic primary BC cell line, DT28. We also employed the MMTV-PyMT spontaneous model of BC metastasis to evaluate cCAFs and CTCs in a preclinical model. Mice were sacrificed at specific time points, and cardiac blood was collected to ascertain the temporal dynamics of cCAF and CTC presence. Blood was filtered using the faCTChecker microfluidic filtration instrument (Circulogix). Filters were enumerated by IF for cCAFs, CTCs, and cCAF/CTC co-clusters. To explore BC-intrinsic factors that influence cCAF and cCAF/CTC cluster egress, we modeled egress from the primary tumor in vitro using transendothelial cell migration assays, where the chemoattractant for CAF cells was conditioned media from the different BC cells lines. Results: In both spontaneous and orthotopic xenograft models of BC, cCAFs, CTCs, and cCAF/CTCs appear early in tumor development. cCAF/CTC clusters increase in correlation with tumor burden and metastasis. CAFs injected alone in orthotopic xenografts are able to egress independently of BC cells and cCAFs and cCAF clusters were seen; however, co-inoculation with BC cells resulted in substantially higher numbers of both individual cCAFs and cCAF clusters, and now co-clusters were seen. cCAFs appear about 4 days post-injection, and precede appearance of CTCs and CAF/CTC clusters. CAFs co-injected with BC cells into the MFP appear at metastatic sites. In in vitro transendothelial assays, BC secreted factors that potentiate CAF egress are identified as well as the if cCAF/CTC clusters egress as clusters or cluster in the circulation. Conclusion: Although CAFs are highly motile and cCAFs precede CTCs into the circulation, BC subtype influences the ability of CAFs to egress. Targeting cCAF egress and/or cCAF/BC cell clusters provides novel avenues to prevent or treat BC metastasis. Citation Format: Utsav Sharma, Philip Miller, Kelsie Medina Saenz, Angela Spartz, Marc Lippman, Dorraya El-Ashry. CAFs in breast cancer circulation: The influence of cancer cell intrinsic mechanisms [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 5062.

Background: Cancer is one of the major causes of the death and affects people of all ages throughout the world. The drugs that are currently available to treat cancer have many side effects. Hence, there is considerable scientific interest in the continuing discovery of new anticancer agents from natural sources. The aim of this study was to prepare and characterize nanoparticles combining Indigofera intricata crude alcoholic extract and chitosan and to evaluate the anticancer cell proliferative activity for both extract and nanoparticles. Methods: Dried alcoholic extract was prepared and characterized for its phenolic and flavonoid contents. Chitosan extract nanoparticles was prepared by ionic gelation method and characterized by thin layer chromatography (TLC), Fourier-transform infrared spectroscopy (FTIR), particle size and zeta-potential analysis. The anticancer cell proliferative activities of both plant extract and nanoparticles at different concentrations were evaluated using breast cancer cell line (MCF 7). Results: The alcoholic extract showed high contents from both phenolic and flavonoid constituents (15 % and 22 % respectively). The interaction of polyphenolic compounds of the extract with chitosan was confirmed by the TLC and FTIR results. The particle size and zeta-potential of nanoparticles found to be 400.6nm ± 101.8 nm and +42.1 mV ± 9.27 mV respectively. The plant extract showed the lowest cell viability of 45.21% ± 4.8% at the highest dose (250 mg) tested in this investigation. Almost 500-fold reduction (from 250 mg to 0.5 mg) in the extract concentration required to achieve same anticancer cell proliferative activity when formulated as nanoparticles. Also 2.5 mg extract containing nanoparticles showed similar anticancer cell proliferative activity as 5 mg 5-FU. Conclusion: Our results revealed that traditional medicinal plants could be an excellent source of natural anticancer agents and the chitosan-extract nanoparticles is a promising formulation strategy to enhance their clinical effectiveness.

Characterization and carboplatin loaded chitosan nanoparticles for the chemotherapy against breast cancer in vitro studies

Embedding natural products into chitosan nanoparticles (CNP) is an effective way to produce a novel combination with better antimicrobial and anticancer activities. Therefore, this study aims to incorporate carob honey (CH) into CNP, determine its potential antimicrobial along with antiproliferative activities, by well diffusion and MTT cell viability assays, respectively. Successful loading of CH in CNP was confirmed after due characterization. The nanoparticles, synthesized by ionic gelation method, produced a small (101.3 ± 4.13 nm), stable (+27.27 ± 0.95 mV), and monodispersed (0.2265 ± 0.0027) CH-loaded CNP (CHCNP). The best antibacterial activity occurred in Klebsiella pneumoniae (K. pneumoniae) (23 ± 0 mm to 16 ± 1.7 mm) followed by Escherichia coli (E. coli) (18 ± 2.0 mm to 10 ± 1 mm). Meanwhile, Aspergillus niger (A. niger) and Aspergillus flavus (A. flavus) were evenly inhibited with inhibition zones in the range of 15 ± 3 mm to 7 ± 0.8 mm and 15 ± 5 mm to 9 ± 1.4 mm, respectively. CHCNP showed a remarkable cytotoxic effect on MDA-MB-231 according to concentration and time, with IC50 of 25 ± 5 to 18 ± 2.6 μg/mL within 24–72 h. These findings demonstrated the feasibility of loading CH in CNP to form a nanoformulation that could potentially serve as a target-specific therapeutic agent in the treatments of microbial infections and breast cancer. However, there is a need for further research on the safety, dosage optimization, in vivo studies and mechanisms of action of the nanoparticles.

BackgroundThe encapsulation of plant essential oils (EOs) with polymeric materials (e.g. chitosan (CS) and N, N, N-trimethyl chitosan (TMC)) and the further reduction of the polymers into their nano sizes are gaining research interest in nanotechnology due to potential applications in medical drug delivery systems as well as the food and pharmaceutical industry. The present study reports a novel approach for the synthesis of Ocimum gratissimum essential oil (OGEO)-loaded CS and TMC nanoparticles with distinct bioactive and physiochemical properties.MethodsThe OGEO-loaded CS and TMC nanoparticles were characterised using various microscopic and spectroscopic techniques. The bioactive compounds in Ocimum gratissimum methanolic extract (OG-MeOH) and EOs was evinced with gas chromatography-mass spectrometry (GC-MS). Total phenolic content (TPC) of OGEO and OG-MeOH was determined using the Folin-Ciocalteu method. The in vitro drug release kinetic pattern was ascertained by membrane dialysis, while antioxidant activity was determined by the 2,2-diphenyl-1picrylhydrozyl (DPPH) free radical scavenging method. The disc diffusion method was used for antibacterial activity evaluation, while MTT and a trypan blue dye exclusion assay were used to assess cytotoxic activity on MDA-MB-231 breast cancer cells.ResultsGC-MS analysis revealed components that have not been previously reported for Ocimum gratissimum. The maximum OGEO cumulative drug release percentage in vitro was observed at pH 3 for both OGEO-loaded chitosan nanoparticles (OGEO-CSNPs) and OGEO-loaded N, N, N-trimethyl chitosan nanoparticles (OGEO-TMCNPs). The antioxidant activity of OGEO-CSNPs and OGEO-TMCNPs never reached a steady state after 75 h. OGEO-TMCNPs exhibited antibacterial activity at a lower concentration for both Gram-negative and Gram-positive food pathogens. In vitro cytotoxicity revealed the increased toxicity of OGEO-TMCNPs on MDA-MB-231 breast cancer cell lines.ConclusionOGEO-loaded CS and TMC nanoparticles were synthesised using a novel material optimisation approach. The synthesised nanoparticles have shown a promising application in the pharmaceutical and food industries.

Breast cancer is one of cancer causes of death in woman. Chemotherapy is one cancer treatment give toxic effects on normal cells. Alternative of cancer treatment by using flavonoid derivative have potent anticancer to reduce side effects of cancer. Chalcone is family of flavonoid that have biological activity. Chalcone derivatives have potential compound as anticancer agent. Chalcone with the presence halogen, metoxy group in ring B is know to inhibit cancer cells. The aims of this research were to synthesize chalcone derivate with bromo, methoxy, and hyroxy group in ring chalcone and to determine the anticancer activity of chalcone derivative. The chalcone derivative was synthesized from 2-hydroxyacetophenone with 2-bromo-4,5-dimethoxybenzaldehyde by Claisen-Schmidt reaction. In vitro cytotoxicity against breast cancer cell was tested by MTT assay method. The compound of 2’-hydroxy-2-bromo-4,5-dimethoxychalcone was yield in 78% as yellow solid. The IC50 of 2’-hydroxy-2-bromo-4,5-dimethoxychalcone was 42,19 µg/mL as a moderate activity to inhibiting breast cancer cell line. Cytotoxity of docorubicin againts breast cancer cell line more active than 2’-hydroxy-2-bromo-4,5-dimethoxychalcone with IC50 10,61 µg/mL. Doxorubicin as drug standar had better anticancer activity than 2’-hydroxy-2-bromo-4,5-dimethoxychalcone. Based on the IC50 value, the compound 2’-hydroxy-2-bromo-4,5-dimethoxychalcone has a moderate activity towards breast cancer cell lines. This study can recommend as candidate for anticancer againts breast cancer cell lines.