Doxorubicin-Mediated Bone Loss in Breast Cancer Bone Metastases Is Driven by an Interplay between Oxidative Stress and Induction of TGFβ

<div><p>Breast cancer patients, who are already at increased risk of developing bone metastases and osteolytic bone damage, are often treated with doxorubicin. Unfortunately, doxorubicin has been reported to induce damage to bone. Moreover, we have previously reported that doxorubicin treatment increases circulating levels of TGFβ in murine pre-clinical models. TGFβ has been implicated in promoting osteolytic bone damage, a consequence of increased osteoclast-mediated resorption and suppression of osteoblast differentiation. Therefore, we hypothesized that in a preclinical breast cancer bone metastasis model, administration of doxorubicin would accelerate bone loss in a TGFβ-mediated manner. Administration of doxorubicin to 4T1 tumor-bearing mice produced an eightfold increase in osteolytic lesion areas compared untreated tumor-bearing mice (<i>P</i> = 0.002) and an almost 50% decrease in trabecular bone volume expressed in BV/TV (<i>P</i> = 0.0005), both of which were rescued by anti-TGFβ antibody (1D11). Doxorubicin, which is a known inducer of oxidative stress, decreased osteoblast survival and differentiation, which was rescued by N-acetyl cysteine (NAC). Furthermore, doxorubicin treatment decreased Cu-ZnSOD (SOD1) expression and enzyme activity <i>in vitro</i>, and treatment with anti-TGFβ antibody was able to rescue both. In conclusion, a combination therapy using doxorubicin and anti-TGFβ antibody might be beneficial for preventing therapy-related bone loss in cancer patients.</p></div

Doxorubicin mediates bone loss by elevating oxidative stress.

<p>(A) C400 oxidation show increase in reactive oxygen species (ROS) upon <i>in vitro</i> doxorubicin (0.01 ug/ml, 20 hours) treatment increases oxidative stress in the mouse bone marrow stromal cells, which was decreased by concomitant treatment with 1D11(25 µg/ml). Data represents average percentage of C400 positive cells from triplicate samples. (B) RT-PCR showing a decrease in SOD1 (copper zinc superoxide dismutase 1) and GPx expression was noted in MC3T3 mouse osteoblast cells upon treatment with doxorubicin (0.01 µg/ml, 20 hours), which was returned to normal level by co-treatment with anti-TGFβ antibody 1D11(25 ug/ml). (C) SOD1 expression normalized against GAPDH expression, quantified by Image J. (D) GPx expression normalized against GAPDH expression, quantified by Image J. (E) SOD1 activity was performed using MC3T3 cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a> section. The inhibition of SOD1 activity was measured by formation of NBT-diformazan from NBT following 20 hours treatment in either serum free alpha-MEM media alone, or supplemented with 0.01 ug/ml doxorubicin, 25 µg/ml anti-TGFβ antibody and a combination of both doxorubicin and anti-TGFβ antibody. A drastic inhibition of SOD1 activity was noted following doxorubicin treatment which was restored by anti-TGFβ antibody. (F) Calvarial osteoblasts from wild type mice (3–4 days old pups) were cultured until confluent and treated with osteoblast differentiation media supplemented with doxorubicin (0.01 µg/ml), N-acetyl cysteine (NAC, 20 mM) treatment, or a combination of both, or media alone until mineralized matrix was formed. Quantification of Von Kossa staining images from at least 3 different fields were done using Metamorph software.</p

Anti-TGFβ antibody inhibits doxorubicin-mediated increase in osteoclast formation.

<p>Mononuclear cells from spleen (A) and bone marrow (B) from C57BL/6 were isolated and cultured for 15 days in the presence of MCSF and RANKL until mature osteoclasts are formed and scored using TRAP staining as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a>. (C) Osteoblast-mediated osteoclast formation was also done using a co-culture system as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a>. Student T-test was performed and P<0.05 was considered significant.</p

Doxorubicin treatment accelerates bone loss in preclinical breast cancer bone metastasis model.

<p>Five-week old female Balb/C mice received either 5 mg/Kg doxorubicin (i.p. once weekly for three weeks) or PBS and microCT of tibiae were performed. For orthotopic breast cancer bone metastasis model, another set of four-week old female Balb/C mice were injected in the left #4 mammary fat pad using a bone-tropic 4T1 cell line and upon tumor development (palpable size after 1 week) mice were treated with either PBS or doxorubicin (5 mg/Kg doxorubicin, i.p., once weekly for three weeks) and microCT and histology of tibiae were performed. (A) Schematic representation of orthotopic injection of 4T1 cells in mammary fat pad. (B) Histology of mouse tibia showing tumor cells in the bone. (C) Representative microCT images (3D reconstruction) of tibiae collected from non-tumor bearing and tumor-bearing Balb/C mice after three weeks of treatment and (D) Quantification of average BV/TV showing further decrease in trabecular bone volume in tumor-bearing mice upon doxorubicin treatment, compared to non-tumor bearing mice. (E) Average BV/TV were assessed using microCT analysis of tibiae collected from 4 week old athymic nude mice mouse treated with either vehicle or doxorubicin showing loss of trabecular bone volume (<i>P</i> = 0.02). (F) Representative X-ray images showing bone loss upon doxorubicin treatment, compared to control. Quantification of (G) osteolytic lesion area and (H) osteolytic lesion numbers in tumor-bearing Balb/C mice receiving 4T1 cell via cardiac injection and treated with either PBS or doxorubicin (5 mg/Kg) once per week for three weeks. (I) Ratlaps ELISA showing significant increase (<i>P</i><0.05) in bone resorption upon doxorubicin treatment in non-tumor bearing mice. Statistical analysis was performed using student’s T–test. <i>P</i><0.05 was considered significant. At least 5 mice were used in each group for these experiments.</p

Anti-TGFβ antibody rescues doxorubicin mediated bone loss in breast cancer bone metastasis.

<p>MDA-MB-231 cells (1×10⁶) were inoculated via left cardiac ventricle of four-week old female athymic nude mice and treated with PBS, doxorubicin (5 mg/Kg, once weekly for 4 weeks, i.p.), 1D11 (10 mg/Kg, three times per weekly for 4 weeks) and a combination of doxorubicin and 1D11 for three weeks. (A) Representative microCT images of mice tibiae from each treatment group (B) Quantification of trabecular bone volume (BV/TV), (C) Histology of mice tibia from each group revealed trabecular bone loss upon doxorubicin treatment, both of which were rescued upon anti-TGFβ antibody treatment. Quantification of (D) average trabecular numbers and (E) average trabecular thickness. Representative microCT images from tumor-bearing mice in which (F) 4T1 cells (10⁵) were inoculated either via intracardiac route or (H) in the #4 mammary fat pad of four-week old female Balb/c mice which received either PBS, doxorubicin (5 mg/Kg, once weekly for 4 weeks, i.p.), 1D11 (10 mg/Kg, three times per weekly for 4 weeks) or a combination of doxorubicin and 1D11 for three weeks. Quantification of microCT images show significant loss of trabecular bone volume (BV/TV) in both intracardiac (G) and orthotopic (I) models. At least 5 mice were assessed in each group and P<0.05 was considered significant.</p

Anti-TGFβ antibody treatment improves doxorubicin-mediated inhibition of osteoblast differentiation and increases the frequency of osteoblast colony forming units.

<p>Mouse bone marrow cells were flushed and allowed to attach for two days. Bone marrow stromal cells were trypsinized and replated as 1×10⁶ cells per well in six well plates for fibroblast colony forming units (CFU-F) and 2×10⁶ cells for osteoblast colony forming units (CFU-OB). Cells were cultured either using DMEM-F12 media (10% FBS) alone or supplemented with either doxorubicin (0.01 ug/ml), 1D11(25 µg/ml) or a combination of both until fibroblast colonies were formed. CFU-OB were cultured using osteoblast differentiation media (alpha-MEM+10% FBS) containing ascorbic acid and β glycerophosphate with similar concentration of doxorubicin (0.01 µg/ml) and/or 1D11(25 µg/ml). Upon microscopic colony formation, media were aspirated, plates were washed in PBS, fixed with 10% neutral buffered formalin and stained to score (A) Average number of fibroblast colony forming units (CFU-F) per 1×10⁶ bone marrow cells. (B) Average number of osteoblast colony forming units (CFU-OB) per 2×10⁶ bone marrow cells. (C) Ex <i>vivo</i> osteoblast mineralization assay was performed using mouse calverial osteoblasts isolated from 3 days old pups and plated in triplicate. Upon confluence, cells were grown in osteoblast differentiation media containing ascorbic acid and β glycerophosphate, in presence of doxorubicin (0.01 µg/ml), 1D11(25 µg/ml) or a combination until mineralized matrix were formed. Von Kossa staining was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078043#s4" target="_blank">Materials and Methods</a> and mineralization was scored using Metamorph software in each set and compared with media only group. Student T-test was performed to calculate p-values. P>0.05 was considered significant. N = 6 for each group was used in this experiment. (D) RT-PCR for expression of RANKL, OPG, OCN and OPN from MC3T3 cells treated with media alone, doxorubicin (0.01 µg/ml, 20 hours), anti-TGFβ antibody (25 ug/ml) and a combination of doxorubicin and 1D11.</p

Octa-arginine modified poly(amidoamine) dendrimers for improved delivery and cytotoxic effect of paclitaxel in cancer

<p>Cell penetrating peptides (CPP) have the ability to penetrate the cell membrane and have been associated with various cargos for their facile intracellular translocation. The current study involves the synthesis of a CPP, octa-arginine (R8)-modified poly(amidoamine) dendrimer of generation 4 (G4), which has additionally been PEGylated and conjugated to the poorly soluble anticancer drug, paclitaxel (PTX). The synthesized dendrimer conjugates were characterized by proton nuclear magnetic resonance (1H-NMR) Spectroscopy and zeta potential measurements and evaluated <i>in vitro</i> in cell monolayers and 3D spheroids. Cellular uptake study in human cervical cancer cell line (HeLa) revealed that R8 modification significantly improved the cell association of conjugates. G4-PTX- polyethylene glycol (PEG)-R8 conjugate demonstrated enhanced cytotoxic potential and higher induction of apoptosis compared to free PTX and G4-PTX-PEG. Further, the penetrability of fluorescently labeled F-G4-PTX-PEG-R8 was evaluated in 3D spheroids of HeLa at various depths by using confocal microscopy. G4-PTX-PEG-R8 induced cell death and inhibited the growth in 3D spheroids as competently as in monolayers. The enhanced intracellular translocation of R8-modified dendrimers resulted in improved anticancer efficacy of PTX. Therefore, the newly developed dendrimer system is efficient for the intracellular delivery of PTX in cancer cells and has a strong potential to be utilized as an effective chemotherapeutic agent for cancer.</p

GH1: GH scores with skipped pedigrees; GH2: GH scores without skipped pedigrees

<p><b>Copyright information:</b></p><p>Taken from "Linkage analysis of the simulated data – evaluations and comparisons of methods"</p><p>http://www.biomedcentral.com/1471-2156/4/s1/S70</p><p>BMC Genetics 2003;4(Suppl 1):S70-S70.</p><p>Published online 31 Dec 2003</p><p>PMCID:PMC1866509.</p><p></p

Polylactide-Based Block Copolymeric Micelles Loaded with Chlorin e6 for Photodynamic Therapy: <i>In Vitro</i> Evaluation in Monolayer and 3D Spheroid Models

Recently, photodynamic therapy (PDT) has found wide application as a noninvasive treatment modality for several cancers. However, the suboptimal delivery of photosensitizers (PSs) to the tumor site is a drawback, which inhibits the effectiveness of PDT. Hydrophobicity, strong oxygen and light dependence, and limited tissue penetrability of photosensitizers represent the major barriers to the clinical application of PDT. In order to improve biopharmaceutical properties of a clinically approved photosensitizer chlorin e6 (Ce6), we developed a nanoformulation encapsulating Ce6 in methoxy-poly­(ethylene glycol)-poly­(d,l-lactide) (mPEG-PLA) copolymeric micelles. The physicochemical properties, including particle size, zeta potential, encapsulation efficiency, drug loading, generation of reactive oxygen species following near-infrared light illumination (633 nm), and <i>in vitro</i> drug release, were determined. The therapeutic efficacy of Ce6-mPEG-PLA micelles following illumination were evaluated <i>in vitro</i> in both two- and three-dimensional cell culture systems by using human uterine cervix carcinoma (HeLa) and human alveolar adenocarcinoma (A549) cells in monolayers and in A549 spheroids, respectively. The mPEG-PLA micelles were stable with a particle size of 189.6 ± 14.32 nm and loaded Ce6 efficiently (encapsulation efficiency ∼75%). The Ce6-loaded micelles generated singlet oxygen at a higher concentration compared to free Ce6 in aqueous media. Ce6-mPEG-PLA micelle mediated PDT showed improved cellular internalization in both of the cell lines, resulting in enhanced cytotoxicity compared to free Ce6. In contrast, the Ce6-loaded micelles did not show any cytotoxicity in the absence of irradiation. The Ce6-loaded micelles exhibited deep penetration in the spheroids leading to phototoxicity and cellular apoptosis in the A549 spheroidal model. Results from this study indicated that the newly developed nanoformulation of Ce6 could be utilized in PDT as an effective treatment modality for solid tumors

Transferrin and octaarginine modified dual-functional liposomes with improved cancer cell targeting and enhanced intracellular delivery for the treatment of ovarian cancer

<p>Off-target effects of drugs severely limit cancer therapy. Targeted nanocarriers are promising to enhance the delivery of therapeutics to tumors. Among many approaches for active tumor-targeting, arginine-rich cell penetrating peptides (AR-CPP) and ligands specific to target over-expressed receptors on cancer-cell surfaces, are popular. Earlier, we showed that the attachment of an AR-CPP octaarginine (R8) to the surface of DOXIL^® (Doxorubicin encapsulated PEGylated liposomes) improved cytoplasmic and nuclear DOX delivery that enhanced the cytotoxic effect <i>in vitro</i> and improved therapeutic efficacy <i>in vivo</i>. Here, we report on DOX-loaded liposomes, surface-modified with, R8 and transferrin (Tf) (Dual DOX-L), to improve targeting of A2780 ovarian carcinoma cells via the over-expressed transferrin receptors (TfRs) with R8-mediated intracellular DOX delivery. Flow cytometry analysis with fluorescently labeled DualL (without DOX) showed two-fold higher cancer-cell association than other treatments after 4 h treatment. Blocking entry pathways of R8 (macropinocytosis) and Tf (receptor-mediated endocytosis, RME) resulted in a decreased cancer-cell association of DualL. Confocal microscopy confirmed involvement of both entry pathways and cytoplasmic liposome accumulation with nuclear DOX delivery for Dual DOX-L. Dual DOX-L exhibited enhanced cytotoxicity <i>in vitro</i> and was most effective in controlling tumor growth <i>in vivo</i> in an A2780 ovarian xenograft model compared to other treatments. A pilot biodistribution study showed improved DOX accumulation in tumors after Dual DOX-L treatment. All results collectively presented a clear advantage of the R8 and Tf combination to elevate the therapeutic potential of DOX-L by exploiting TfR over-expression imparting specificity followed by endosomal escape and intracellular delivery via R8.</p