Investigation of NPB Analogs That Target Phosphorylation of BAD-Ser99 in Human Mammary Carcinoma Cells

The design and development of a small molecule named NPB [3-{(4(2,3-dichlorophenyl)piperazin-1-yl}{2-hydroxyphenyl)methyl}-N-cyclopentylbenzamide], which specifically inhibited the phosphorylation of BAD at Ser99 in human carcinoma cells has been previously reported. Herein, the synthesis, characterization, and effect on cancer cell viability of NPB analogs, and the single-crystal X-ray crystallographic studies of an example compound (4r), which was grown via slow-solvent evaporation technique is reported. Screening for loss of viability in mammary carcinoma cells revealed that compounds such as 2[(4(2,3-dichlorophenyl)piperazin-1-yl][naphthalen-1-yl]methyl)phenol (4e), 5[(4(2,3-dichlorophenyl)piperazin-1-yl][2-hydroxyphenyl)methyl)uran-2-carbaldehyde (4f), 3[(2-hydroxyphenyl][4(p-tolyl)piperazin-1-yl)methyl)benzaldehyde (4i), and NPB inhibited the viability of MCF-7 cells with IC50 values of 5.90, 3.11, 7.68, and 6.5 µM, respectively. The loss of cell viability was enhanced by the NPB analogs synthesized by adding newer rings such as naphthalene and furan-2-carbaldehyde in place of N-cyclopentyl-benzamide of NPB. Furthermore, these compounds decreased Ser99 phosphorylation of hBAD. Additional in silico density functional theory calculations suggested possibilities for other analogs of NPB that may be more suitable for further development.

In vitro and in vivo analysis of microvesicle-mediated metastasis using a bright, red-shifted bioluminescent reporter protein of extracellular vesicles

Cancer cells produce heterogeneous extracellular vesicles (EVs) as mediators of intercellular communication. Our study focused on a novel method to image EV subtypes and their biodistribution in vivo. Regardless of injection routes, we established that reporter EVs isolated from murine mammary carcinoma cells expressing PalmReNL, which utilizes bioluminescence resonance energy transfer (BRET), localized to the lungs. This new EV reporter allowed highly sensitive EV tracking in vitro and in vivo and enabled us to begin studies to understand the commonalities and functional differences of the EV subtypes. We demonstrated the early appearance of metastatic foci in the lungs of mammary tumor-bearing mice following multiple injections of the microvesicle (MV)-enriched fraction derived from mammary carcinoma cells. In addition, the results we present here show that tumor cell-derived MVs act on distant tissues through upregulating LC3 expression within the lung.

Involvement of Nestin in the Progression of Canine Mammary Carcinoma

Nestin, a class VI intermediate filament protein, is known to be expressed in various types of human neoplasms, including breast cancer, and is associated with their progression. However, its expression and role in canine mammary tumors remain unknown. We analyzed nestin expression in canine mammary tumors using in situ hybridization and immunohistochemistry. We also investigated its role in a canine mammary carcinoma cell line using RNA interference. Nestin expression was not observed in luminal epithelial cells of any of the 62 cases of benign mammary lesions examined, although myoepithelial cells showed its expression in most cases. In 16/50 (32%) primary mammary carcinomas and 6/15 (40%) metastases of mammary carcinomas, cytoplasmic nestin expression was detected in luminal epithelial cells. In luminal cells of primary mammary carcinomas, its expression was positively related to several pathological parameters that indicate high-grade malignancy, including histological grading ( P < .01), vascular/lymphatic invasion ( P < .01), Ki-67 index ( P < .01), and metastasis ( P < .05). Immunohistochemistry revealed that nestin expression was related to vimentin expression in mammary carcinomas ( P < .01). This relationship was confirmed using reverse transcription-quantitative polymerase chain reaction using 9 cell lines derived from canine mammary carcinoma ( P < .01). Finally, nestin knockdown in canine mammary carcinoma cells using small interfering RNA inhibited cell proliferation and migration based on WST-8, Boyden chamber, and cell-tracking assays. These findings suggest that nestin may at least partially mediate these behaviors of canine mammary carcinoma cells.

Combined Ultrasound Treatment with Transferrin-Coupled Nanoparticles Improves Active Targeting of 4T1 Mammary Carcinoma Cells

Objective: Conventional chemotherapy remains the mainstay treatment for many breast cancer patients, but its effectiveness is limited by toxic side effects. Incorporating drugs such as docetaxel into nanoparticle medicines can reduce toxicity but further improvements are required. To facilitate more active tumor targeting, we prepared transferrin-docetaxel-loaded pegylated-albumin nanoparticles (Tf-PEG-DANPS). Methods: The growth inhibitory effects and the ability of unmodified DANPS or PEG-DANPS to induce apoptosis in 4T1 mouse mammary cancers were compared to Tf-PEG-DANPS treatment using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and flow cytometry. These experiments were extended in vivo to the intravenous treatment of 4T1 tumors where PEG-DANPS was compared to Tf-PEG-DANPS alone or Tf-PEG-DANPS combined with ultrasound (US + Tf-PEG-DANPS). Histological assessments using hematoxylin and eosin (HE) sections were performed to examine antitumor activity, metastasis to lung and liver, and body weight measurements taken as an indicator of toxicity. Results: MTT experiments show that, in the normal and low concentration interval, the inhibition ability of the Tf-PEG-DANPS is higher than that of other drug-giving groups, and the flow cytometry show that the proportion of induced apoptosis in each given group is 2.88%, 42.95%, 48.23%, and 57.89%, indicating that the Tf-PEG-DANPS group has more significant ability to induce apoptosis than other drug-giving groups. From the pathological HE staining and semiquantitative analysis, US+Tf-PEG-DANPS can effectively inhibit the growth of breast cancer transplanted tumors and suppress metastases, it also has smaller toxic side effects on mice. Conclusion: The antitumor effect of US+Tf-PEG-DANPS represents an effective combination that exhibits increased antitumor activity and metastasis reduction with an improved side-effect profile.

Environmental control of mammary carcinoma cell expansion by acidification and spheroid formation in vitro

Breast cancer is the leading cause of cancer death among women worldwide. Like other cancers, mammary carcinoma progression involves acidification of the tumor microenvironment, which is an important factor for cancer detection and treatment strategies. However, the effects of acidity on mammary carcinoma cell morphology and phenotype have not been thoroughly characterized. Here, we evaluated fundamental effects of environmental acidification on mammary carcinoma cells in standard two-dimensional cultures and three-dimensional spheroids. Acidification decreased overall mammary carcinoma cell viability, while increasing their resistance to the anthracycline doxorubicin. Environmental acidification also increased extracellular vesicle production by mammary carcinoma cells. Conditioned media containing these vesicles appeared to increase fibroblast motility. Acidification also increased mammary carcinoma cell motility when cultured with fibroblasts in spheroids. Taken together, results from this study suggest that environmental acidification induces drug resistance and extracellular vesicle production by mammary carcinoma cells that promote tumor expansion.