Nanoparticle-Based Drug Delivery Systems for Cancer Therapy: Formulation and In-Vitro Evaluation

Genes encoding growth-inhibitory proteins are postulated to be candidate tumor suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumors. Here we report the cloning and functional characterization of a novel human bone marrow stromal cell (BMSC)-derived growth inhibitor (BDGI) by large scale random sequencing of a human BMSC cDNA library. Human BDGI cDNA encodes a 477-amino acid residue protein that shares high homology with rat and mouse pregnancy-induced growth inhibitors. The C-terminal of BDGI is identical to a novel human pregnancy-induced growth inhibitor, OKL38. BDGI is also closely related to many other eukaryotic proteins, which together form a novel and highly conserved family of BDGI-like proteins. BDGI overexpression inhibits the proliferation, decreases anchorage-dependent growth, and reduces migration of MCF-7 human breast cancer cells, whereas down-regulation of BDGI expression promotes the proliferation of MCF-7 and HeLa cervix epitheloid carcinoma cells. Interestingly, the inhibitory effect of BDGI on MCF-7 cells is more potent than that of OKL38. We demonstrate that BDGI induces cell cycle arrest in S phase and subsequent apoptosis of MCF-7 cells, which is likely to account for the antiproliferative effects of BDGI. This process may involve up-regulation of p27Kip1 and down-regulation of cyclin A, Bcl-2, and Bcl-xL. The inhibitory effect of BDGI on cell proliferation and the induction of apoptosis were also observed in A549 lung cancer cells but not HeLa cells. These results indicate that BDGI might be a growth inhibitor for human tumor cells, especially breast cancer cells, possibly contributing to the development of new therapeutic strategies for breast cancer.

ATF5 loss of function has been shown previously to cause apoptotic cell death in glioblastoma and breast cancer cells but not in non-transformed astrocytes and human breast epithelial cells. The mechanism for the cell type-dependent survival function of ATF5 is unknown. We report here that the anti-apoptotic factor BCL-2 is a downstream target of ATF5 that mediates the prosurvival function of ATF5 in C6 glioma cells and MCF-7 breast cancer cells. ATF5 binds to an ATF5-specific regulatory element that is downstream of and adjacent to the negative regulatory element in the BCL-2 P2 promoter, stimulating BCL-2 expression. Highlighting the critical role of BCL-2 in ATF5-dependent cancer cell survival, expression of BCL-2 blocks death of C6 and MCF-7 cells induced by dominant-negative ATF5, and depletion of BCL-2 impairs ATF5-promoted cell survival. Moreover, we found that BCL-2 expression is not regulated by ATF5 in non-transformed rat astrocytes, mouse embryonic fibroblasts, and human breast epithelial cells, where expression of BCL-2 but not ATF5 is required for cell survival. These findings identify BCL-2 as an essential mediator for the cancer-specific cell survival function of ATF5 in glioblastoma and breast cancer cells and provide direct evidence that the cell type-specific function of ATF5 derives from differential regulation of downstream targets by ATF5 in different types of cells.

Aberrant activity of the phosphatidylinositol 3-kinase (PI3K) pathway supports growth of many tumors including those of breast, lung, and prostate. Resistance of breast cancer cells to targeted chemotherapies including tyrosine kinase inhibitors (TKI) has been linked to persistent PI3K activity, which may in part be due to increased membrane expression of epidermal growth factor (EGF) receptors (HER2 and HER3). Recently we found that proteins of the RGS (regulator of G protein signaling) family suppress PI3K activity downstream of the receptor by sequestering its p85alpha subunit from signaling complexes. Because a substantial percentage of breast tumors have RGS16 mutations and reduced RGS16 protein expression, we investigated the link between regulation of PI3K activity by RGS16 and breast cancer cell growth. RGS16 overexpression in MCF7 breast cancer cells inhibited EGF-induced proliferation and Akt phosphorylation, whereas shRNA-mediated extinction of RGS16 augmented cell growth and resistance to TKI treatment. Exposure to TKI also reduced RGS16 expression in MCF7 and BT474 cell lines. RGS16 bound the amino-terminal SH2 and inter-SH2 domains of p85alpha and inhibited its interaction with the EGF receptor-associated adapter protein Gab1. These results suggest that the loss of RGS16 in some breast tumors enhances PI3K signaling elicited by growth factors and thereby promotes proliferation and TKI evasion downstream of HER activation.

Recently dynein light chain 1 (DLC1), a cytoskeleton signaling component, has been shown to interact with and transactivate estrogen receptor-alpha (ER), leading to increased expression of ER target genes and growth stimulation of breast cancer cells. However, the molecular mechanism by which DLC1 regulates the ER pathway remains poorly understood. To gain insights into the putative mechanism, here we set out to identify novel DLC1-interacting proteins. We identified KIBRA, a WW domain- and a glutamic acid stretch-containing protein, as a DLC1-binding protein and showed that it interacts with DLC1 both in vitro and in vivo. We found that KIBRA-DLC1 complex is recruited to ER-responsive promoters. We also found that KIBRA-DLC1 interaction is mandatory for the recruitment and transactivation functions of ER or DLC1 to the target chromatin. Finally we found that KIBRA interacts with histone H3 via its glutamic acid-rich region and that such interaction might play a mechanistic role in conferring an optimal ER transactivation function as well as the proliferation of ligand-stimulated breast cancer cells. Together these findings indicate that DLC1-KIBRA interaction is essential for ER transactivation in breast cancer cells.

Activation of p38 MAPK is a key pathway for cell proliferation and differentiation in breast cancer and thyroid cells. The sodium/iodide symporter (NIS) concentrates iodide in the thyroid and lactating breast. All-trans-retinoic acid (tRA) markedly induces NIS activity in some breast cancer cell lines and promotes uptake of β-emitting radioiodide (131)I sufficient for targeted cytotoxicity. To identify a signal transduction pathway that selectively stimulates NIS expression, we investigated regulation by the Rac1-p38 signaling pathway in MCF-7 breast cancer cells and compared it with regulation in FRTL-5 rat thyroid cells. Loss of function experiments with pharmacologic inhibitors and small interfering RNA, as well as RT-PCR analysis of p38 isoforms, demonstrated the requirement of Rac1, MAPK kinase 3B, and p38β for the full expression of NIS in MCF-7 cells. In contrast, p38α was critical for NIS expression in FRTL-5 cells. Treatment with tRA or overexpression of Rac1 induced the phosphorylation of p38 isoforms, including p38β. A dominant negative mutant of Rac1 abolished tRA-induced phosphorylation in MCF-7 cells. Overexpression of p38β or Rac1 significantly enhanced (1.9- and 3.9-fold, respectively), the tRA-stimulated NIS expression in MCF-7 cells. This study demonstrates differential regulation of NIS by distinct p38 isoforms in breast cancer cells and thyroid cells. Targeting isoform-selective activation of p38 may enhance NIS induction, resulting in higher efficacy of (131)I concentration and treatment of breast cancer.

Hormonal influences on cancer cell proliferation have received much attention with the proposal of autocrine or self-stimulating polypeptide growth factors. The growth of breast cancer is known to be under endocrine control by steroid .hormones. Estrogen stimulates the proliferation of human breast cancer cell lines in vitro and in vivo. Polypeptide growth factors may be common mediators of growth control for both estrogen-regulated and autonomous breast cancer cells. Estrogens induce a large number of enzymes involved in nucleic acid synthesis, the progesterone receptor, plasminogen activator, collagenolytic enzymes, and the laminin receptor. The effect of these proteins includes growth regulation and other activities thought to mediate metastatic events. In addition to the above proteins, growth factors are known to be stimulated by estrogen. It has been observed that the initial growth rate of a hormone-dependent cell line MCF-7 in vitro is proportional to the number of cells plated. This is consistent with the production of autostimulatory growth factors by the MCF-7 cells. More direct studies using estrogen-free extracts of conditioned medium (CM) harvested from MCF-7 cells treated with 17-/%estradiol (E 2) shows that CME 2 stimulated increases in the number of MCF-7 cells. E 2 is an absolute tumor growth requirement for MCF-7 cells. In addition, CME 2 given by continous infusion in vivo to the nude mouse was also capable of stimulating MCF-7 tumorigenesis in the absence of E2. These data support the hypothesis that cultured human breast cancer cells under estrogenic stimulation release a tumor-promoting factor(s) which can act in vitro or in vivo after release into the general circulation of the nude mouse. It has been shown that CM from MCF-7 and other breast cancer cell lines secrete growth-stimulatory activities for both MCF-7 cells and a transforming growth activity for anchorage-independent growth of rodent fibroblasts. Isolation of one of these activities reveals that it coincides with the principal competitor of EGF receptor binding activity. This activity is related to transforming growth factor alpha (TGFe). It has also been shown that this activity is estrogen regulated at the protein and mRNA level. Anti-EGF receptor antibodies decrease the growth of breast cancer cells in culture. It may be that interruption of this loop will be clinically applicable in the future in breast cancer treatment. It has also been shown that insulin-like growth factor-1 (IGF-1) is an autostimulatory mitogen for breast cancer. Breast cancer cells have been shown to secrete a TGF-/~ related activity. TFG-/~ is growth inhibitory for many epithelial and breast cancer cell lines. TGF-/~ is inhibited in MCF-7 cells by treatment with E 2 and conversely stimulated by antiestrogens. Interestingly, TGF-fl inhibits the growth of hormoneindependent cell lines or cells that lack estrogen receptor:. Breast cancers exist as mixtures of estrogen receptor positive and negative tumor cells. Since breast

This study aimed to develop docetaxel (DTX) loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles (DTX-NPs) and to evaluate the different pharmacological sensitivity of NPs to MCF-7 and MDA-MB-231 breast cancer cells. NPs containing DTX or coumarin-6 were prepared by the nanoprecipitation method using PLGA as a polymer and d-α-tocopherol polyethylene glycol 1000 succinate (TPGS) as a surfactant. The physicochemical properties of NPs were characterized. In vitro anticancer effect and cellular uptake were evaluated in breast cancer cells. The particle size and zeta potential of the DTX-NPs were 160.5 ± 3.0 nm and –26.7 ± 0.46 mV, respectively. The encapsulation efficiency and drug loading were 81.3 ± 1.85% and 10.6 ± 0.24%, respectively. The in vitro release of DTX from the DTX-NPs was sustained at pH 7.4 containing 0.5% Tween 80. The viability of MDA-MB-231 and MCF-7 cells with DTX-NPs was 37.5 ± 0.5% and 30.3 ± 1.13%, respectively. The IC50 values of DTX-NPs were 3.92- and 6.75-fold lower than that of DTX for MDA-MB-231 cells and MCF-7 cells, respectively. The cellular uptake of coumarin-6-loaded PLGA-NPs in MCF-7 cells was significantly higher than that in MDA-MB-231 cells. The pharmacological sensitivity in breast cancer cells was higher on MCF-7 cells than on MDA-MB-231 cells. In conclusion, we successfully developed DTX-NPs that showed a great potential for the controlled release of DTX. DTX-NPs are an effective formulation for improving anticancer effect in breast cancer cells.

Ketogenic diets have the potential to lower glucose availability to cancer cells. However, the effect that the resulting increase in ketone bodies has on cancer cells is not fully understood. The present study explored the effect of β-hydroxybutyrate (BHB) on glucose-deprived MCF-7 and T47D breast cancer cells. Cell proliferation was decreased in response to lower glucose conditions, which could not be rescued consistently by 10 or 25 mM BHB supplementation. In addition, gene expression levels were altered when cells were glucose deprived. Reducing glucose availability of cancer cells to 225 mg/l for 4 days significantly decreased the expression of 113 genes and increased the expression of 100 genes in MCF-7 breast cancer cells, and significantly decreased the expression of 425 genes and increased the expression of 447 genes in T47D breast cancer cells. Pathway enrichment analysis demonstrated that glucose deprivation decreased activity of the Hippo-Yap cell signaling pathway in MCF-7 breast cancer cells, whereas it increased the expression of genes in the NRF2-pathaway and genes regulating ferroptosis in T47D breast cancer cells. Treatment of glucose-deprived cells with 10 or 25 mM BHB significantly changed the expression of 14 genes in MCF-7 breast cancer cells and 40 genes in T47D breast cancer cells. No significant pathway enrichment was detected when glucose-deprived cells were treated with BHB. Both cell lines expressed the enzymes (OXCT1/2, BDH1 and ACAT1/2) responsible for metabolizing BHB to acetyl-CoA, yet expression of these enzymes was not altered by either glucose deprivation or BHB treatment. In the publicly available The Cancer Genome Atlas (TCGA), increased expression of ketone body-catabolizing enzymes was observed in various types of cancer based on mRNA expression z-scores. Increased expression of BDH1 and ACAT1 significantly decreased overall survival of patients with breast cancer in TCGA studies, while decreased OXCT1 expression non-significantly decreased overall survival. In conclusion, neither MCF-7 nor T47D breast cancer cells were affected by BHB during glucose deprivation; however, screening of tumors for activation of ketone body-metabolizing enzymes may be able to identify patients that will benefit from ketogenic diet interventions.

Vitamin D(3) compounds are currently in clinical trials for human breast cancer and offer an alternative approach to anti-hormonal therapies for this disease. 1alpha,25-Dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)), the active form of vitamin D(3), induces apoptosis in breast cancer cells and tumors, but the underlying mechanisms are poorly characterized. In these studies, we focused on the role of caspase activation and mitochondrial disruption in 1alpha,25(OH)(2)D(3)-mediated apoptosis in breast cancer cells (MCF-7) in vitro. The effect of 1alpha,25(OH)(2)D(3) on MCF-7 cells was compared with that of tumor necrosis factor alpha, which induces apoptosis via a caspase-dependent pathway. Our major findings are that 1alpha,25(OH)(2)D(3) induces apoptosis in MCF-7 cells by disruption of mitochondrial function, which is associated with Bax translocation to mitochondria, cytochrome c release, and production of reactive oxygen species. Moreover, we show that Bax translocation and mitochondrial disruption do not occur after 1alpha,25(OH)(2)D(3) treatment of a MCF-7 cell clone selected for resistance to 1alpha,25(OH)(2)D(3)-mediated apoptosis. These mitochondrial effects of 1alpha,25(OH)(2)D(3) do not require caspase activation, since they are not blocked by the cell-permeable caspase inhibitor z-Val-Ala-Asp-fluoromethylketone. Although caspase inhibition blocks 1alpha,25(OH)(2)D(3)-mediated events downstream of mitochondria such as poly(ADP-ribose) polymerase cleavage, external display of phosphatidylserine, and DNA fragmentation, MCF-7 cells still execute apoptosis in the presence of z-Val-Ala-Asp-fluoromethylketone, indicating that the commitment to 1alpha,25(OH)(2)D(3)-mediated cell death is caspase-independent.

Nanotechnology-based medical approaches have made tremendous potential for enhancing the treatment efficacy with minimal doses of chemotherapeutic drugs against cancer. In this study, using tamoxifen (Tam), biodegradable antibody conjugated polymeric nanoparticles (NPs) was developed to achieve targeted delivery as well as sustained release of the drug against breast cancer cells. Poly(D,L-lactic-co-glycolic acid) (PLGA) NPs were stabilized by coating with poly(vinyl alcohol) (PVA), and copolymer polyvinyl-pyrrolidone (PVP) was used to conjugate herceptin (antibody) with PLGA NPs for promoting the site-specific intracellular delivery of Tam against HER2 receptor overexpressed breast cancer (MCF-7) cells. The Tam-loaded PVP-PLGA NPs and herceptin-conjugated Tam-loaded PVP-PLGA NPs were characterized in terms of morphology, size, surface charge, and structural chemistry by dynamic light scattering (DLS), Transmission electron microscopy (TEM), ζ potential analysis, 1H nuclear magnetic resonance (NMR), and Fourier transform infrared (FT-IR) spectroscopy. pH-based drug release property and the anticancer activity (in vitro and in vivo models) of the herceptin conjugated polymeric NPs were evaluated by flow cytometry and confocal image analysis. Besides, the extent of cellular uptake of drug via HER2 receptor-mediated endocytosis by herceptin-conjugated Tam-loaded PVP-PLGA NPs was examined. Furthermore, the possible signaling pathway of apoptotic induction in MCF-7 cells was explored by Western blotting, and it was demonstrated that drug-loaded PLGA NPs were capable of inducing apoptosis in a caspase-dependent manner. Hence, this nanocarrier drug delivery system (DDS) not only actively targets a multidrug-resistance (MDR) associated phenotype (HER2 receptor overexpression) but also improves therapeutic efficiency by enhancing the cancer cell targeted delivery and sustained release of therapeutic agents.

Novel Soluplus®—TPGS mixed micelles for encapsulation of paclitaxel with enhanced in vitro cytotoxicity on breast and ovarian cancer cell lines

Recent studies indicate that cancer cells express erythropoietin receptor (EpoR). In this study, we have shown that erythropoietin (Epo) activates the mitogen-activated protein kinase, extracellular signal-regulated kinase (ERK), and promotes migration in MCF-7 breast cancer cells. Epo-stimulated MCF-7 cell migration was blocked by the MEK inhibitor PD098059 and by dominant negative MEK-1, indicating an essential role for ERK. When MCF-7 cells were exposed to hypoxia (1.0% O(2)) for 3 h, the Epo mRNA level increased 2.4 +/- 0.5-fold, the basal level of ERK activation increased, and cell migration increased 2.0 +/- 0.1-fold. Soluble EpoR and Epo-neutralizing antibody significantly inhibited hypoxia-induced MCF-7 cell migration, suggesting a major role for autocrine EpoR cell signaling. MCF-7 cell migration under hypoxic conditions was also inhibited by PD098059. These experiments identify a novel pathway by which exogenously administered Epo, and Epo that is produced locally by cancer cells under hypoxic conditions, may stimulate cancer cell migration.

Platinum-based chemotherapeutic agents are widely employed in cancer treatment because of their effectiveness in targeting DNA. However, this indiscriminate action often affects both cancerous and normal cells, leading to severe side effects and highlighting the need for innovative approaches in achieving precise drug delivery. Nanotechnology presents a promising avenue for addressing these challenges. Protein-based nanocarriers exhibit promising capabilities in the realm of cancer drug delivery with silk sericin nanoparticles standing out as a leading contender. This investigation focuses on creating a sericin-based nanocarrier (SNC) featuring surface charge reversal designed to effectively transport cisplatin (Cispt-SNC) into MCF-7 breast cancer cells. Utilizing AutoDock4.2, our molecular docking analyses identified key amino acids and revealed distinctive conformational clusters, providing insights into the drug-protein interaction landscape and highlighting the potential of sericin as a carrier for controlled drug release. The careful optimization and fabrication of sericin as the carrier material were achieved through flash nanoprecipitation, a straightforward and reproducible method that is devoid of intricate equipment. The physicochemical properties of SNCs and Cispt-SNCs, particularly concerning size, surface charge, and morphology, were evaluated using dynamic light scattering (DLS) and scanning electron microscopy (SEM). Chemical and conformational analyses of the nanocarriers were conducted using Fourier-transform infrared spectroscopy (FTIR) and circular dichroism (CD), and elemental composition analysis was performed through energy-dispersive X-ray spectroscopy (EDX). This approach aimed to achieve the smallest nanoparticle size for Cispt-SNCs (180 nm) and high drug encapsulation efficiency (84%) at an optimal sericin concentration of 0.1% (w/v), maintaining a negative net charge at a physiological pH (7.4). Cellular uptake and cytotoxicity were investigated in MCF-7 breast cancer cells. SNCs demonstrated stability and exhibited a pH-dependent drug release behavior, aligning with the mildly acidic tumor microenvironment (pH 6.0-7.0). Efficient cellular uptake of Cispt-SNC, along with DNA fragmentation and chromatin condensation, was found at pH 6, leading to cell apoptosis. These results collectively indicate the potential of SNCs for achieving controlled drug release in a tumor-specific context. Our in vitro studies reveal the cytotoxicity of both cisplatin and Cispt-SNCs on MCF-7 cells. Cisplatin significantly reduced cell viability at 10 μM concentration (IC50), and the unique combination of sericin and cisplatin showcased enhanced cell viability compared to cisplatin alone, suggesting that controlled drug release is indicated by a gradient decrease in cell viability and highlighting SNCs as promising carriers. The study underscores the promise of protein-based nanocarriers in advancing targeted drug delivery for cancer therapy.

HER2/HER3 regulates extracellular acidification and cell migration through MTK1 (MEKK4)

Small interfering RNA duplexes containing 21-22 nucleotides that mediate sequence-specific mRNA degradation and inhibitory RNA (iRNA) for Sp1 mRNA were used in this study to investigate the role of Sp1 on basal and hormone-induced growth and transactivation in MCF-7 and ZR-75 human breast cancer cells. Transfection of Sp1 iRNA in MCF-7 or ZR-75 cells for 36-44 h decreased Sp1 protein (50-70%) in nuclear extracts, and immunohistochemical analysis showed that the Sp1 protein in transfected MCF-7 cells was barely detectable. In cell cycle progression studies in MCF-7 cells, decreased Sp1 protein was accompanied by a decrease in cells in the S phase and an increase in cells in G(0)/G(1), and estrogen-induced G(0)/G(1) --> S phase progression was inhibited in cells treated with iRNA for Sp1. Sp1 iRNA also specifically blocked basal and estrogen-induced transactivation in cells transfected with a GC-rich construct linked to a luciferase reporter gene (pSp1(3)), and this was accompanied by decreased Sp1 binding to this GC-rich promoter as determined in gel mobility shift and chromatin immunoprecipitation assays. These results clearly demonstrate the key role of the Sp1 protein in basal and estrogen-induced growth and gene expression in breast cancer cells.