Therapy Of Breast Cancer Cells Research Articles

OR04-5 Prohibitin-2 is a binding protein for IGFBP-6 on rhabdomyosarcoma cell membranes and modulates IGFBP-6-dependent migration

to examine whether phosphorylation of IGFBP-3 by DNA-PK is necessary for its apoptotic effect. ATM, which phosphorylates similar sites to DNA-PK, was also investigated. Methods: Two triple-negative breast cancer cell lines (Hs578T and MDAMB468) were treated for 1–48 hours with doxorubicin (0–10 mM), etoposide (0–50 mM) or ionising radiation (0–10 Gy), alone or after pretreatment (30–60 min) with inhibitors of ATM (KU55933; 0–30 mM) or DNA-PK (NU7441; 0–10 mM). DSB were monitored by gH2AX formation, DNA repair processes by kinase autophosphorylation and p53 activation, and apoptosis by caspase-3 activity. Endogenous IGFBP-3, ATM and DNA-PK were downregulated using siRNA. Knockdown (≥80%) was confirmed by qRT-PCR and immunoblot. Results: Although IGFBP3 is reported to be p53-inducible, IGFBP-3 protein was downregulated by doxorubicin treatment in MDAMB468 cells and upregulated in Hs578T cells, even though p53 was activated in both cell lines. This may be due to the different p53 mutations in each cell line. DSB were induced within 1 hour after addition of DNA-damaging agents. DNA damage-stimulated gH2AX and p53 phosphorylation was inhibited by KU55933 but increased by NU7441. DNA damage-induced apoptosis was significantly enhanced in both cell lines by kinase inhibition. In IGFBP-3-silenced cells, doxorubicin-induced apoptosis was significantly attenuated (20%), but neither KU55933 nor NU7441 altered the decreased apoptotic response, suggesting that IGFBP-3 does not need to be phosphorylated by ATM or DNA-PK for its apoptotic effect. In DNA-PK-silenced cells, IGFBP-3 knockdown no longer attenuated doxorubicin-induced apoptosis, suggesting that IGFBP-3 cannot carry out its apoptotic functions in the absence of DNA-PK. Conclusion: We conclude that DNA damaging therapy in breast cancer cells is enhanced by DNA repair inhibitors and is most effective in the presence of both DNA-PK and IGFBP-3. This suggests that cancers positive for IGFBP-3 may be most amenable to this therapy. Further work will employ mass spectrometric methods to determine the specific sites on IGFBP-3 that may become phosphorylated during these treatments. This work was supported by the Australian Research Council (DP0984232) (RCB); and a University of Sydney Postgraduate Award (KAM).

Targeted photodynamic therapy of breast cancer cells using antibody-phthalocyanine-gold nanoparticle conjugates

A 4-component antibody-phthalocyanine-polyethylene glycol-gold nanoparticle conjugate is described for use as a potential drug for targeted photodynamic cancer therapy. Gold nanoparticles (4 nm) were stabilised with a self-assembled layer of a zinc-phthalocyanine derivative (photosensitiser) and a heterobifunctional polyethylene glycol. Anti-HER2 monoclonal antibodies were covalently bound to the nanoparticles via a terminal carboxy moiety on the polyethylene glycol. The nanoparticle conjugates were stable towards aggregation, and under irradiation with visible red light efficiently produced cytotoxic singlet oxygen. Cellular experiments demonstrated that the nanoparticle conjugates selectively target breast cancer cells that overexpress the HER2 epidermal growth factor cell surface receptor, and that they are effective photodynamic therapy agents.

Effective Alloreactive NK Cell Therapy for Breast Cancer In Mice Requires Conditioning with Cyclophosphamide and Total Body Irradiation but Not Bone Marrow Engraftment

AbstractAbstract 2085 Background:Patients with metastasized breast cancer cannot be cured by current standard treatment options. One hypothesis is that slow cycling chemo-resistant tumor stem cells give rise to new tumors after cytoreductive treatment, ultimately leading to chemoresistant tumors. Last year we showed that the 4T1 mouse breast cancer model contains slow-cycling chemo-resistant cells that induce renewed growth of the tumor after chemo- and radiotherapy (abstract 4082). We also showed that haploidentical spleen and bone marrow transplantation (BMT) cures the mice and that donor NK cells are a prerequisite. Our current aim was to study the need of long term BM engraftment and to study the role of the conditioning in the curative process. Methods:The 4T1 breast cancer cell line, originating from a spontaneous Balb/c (H-2d) breast cancer, was cultured under standard conditions. Fifty thousand 4T1 cells were injected s.c. in the flank. For the experiments addressing the need for haploidentical BMT tumor bearing CB6F1 (H-2b/d) recipients were treated with 2x 2Gy total body irradiation and 200 mg/kg cyclophosphamide (CY+TBI) followed by in vitro NK cell enriched haploidentical B6CBAF1 (H-2b/k) spleen cell infusion with or without additional BM cells. Chimerism in tumor-free surviving recipients was measured by flowcytometry of spleens at least 100 days after the treatment. The role of the conditioning in the alloreactive NK cell effect was studied in fully H-2 mismatched B6CBAF1 mice. When indicated, in vivo NK cell depletion was by i.p. injection of anti-AsialoGM1. Results:Figure A shows overall survival of mice with breast cancer after various treatments (10 mice per group). Haploidentical BMT plus spleen cells cured 50% of tumor bearing mice after CY+TBI (♦, dashed line) and survival was at least as good when NK cell enriched spleen cells were co-transplanted (▴, solid line). Transplantation of spleen cells from NK cell depleted mice (•, dotted line) obliterated the beneficial effect of haploidentical transplantation and resulted is similar poor survival as syngeneic BMT plus spleen cells (▪, solid line). The majority of mice that received NK cell enriched spleen cells (10 out of 14 tested) had no bone marrow engraftment and in the other four only 1–5% donor cells were detectable at 150 days. Recipients of unmanipulated haploidentical spleen and BM cells had >90% donor chimerism in 10 out of 14 tested. The cure rate in both groups was nevertheless similarly high. In a subsequent experiment (Figure B, 10 mice per group) we infused haploidentical NK cells only after CY+TBI (▴, solid line); other groups received T cell depleted (x, solid line) or T cell replete (♦, solid line) haploidentical BMT, or syngeneic BMT (▪, solid line). This resulted in a similar superior tumor-free survival (80-90%) than in mice co-transplanted with haploidentical BM (90%), as compared with syngeneic BM and spleen cell transplantation (•, dotted line). [Display omitted] We then planned to study the role of the conditioning in the curative process. For this purpose 4T1 breast cancer cells were injected in fully H-2 mismatched B6CBAF1 mice (H-2b/k). Surprisingly, 4T1 breast cancer is not rejected by B6CBAF1 mice despite the full MHC mismatch. Tumors are only rejected when the mice were treated with CY+TBI. Tumor rejection proved to be NK cell dependant and not a direct result of the conditioning as it was prevented by in vivo NK cell depletion. Conclusions:This report provides the first evidence that chemo resistant tumor cells can be eliminated in vivo by alloreactive NK cells resulting in cure without the need for long term donor bone marrow engraftment. Conditioning with CY+TBI seems essential for this effect. These results set the stage for the exploration of alloreactive NK therapy in patients with metastasized breast cancer. Disclosures:No relevant conflicts of interest to declare.

Hybrid promoters directed tBid gene expression to breast cancer cells by transcriptional targeting

Developing cancer gene therapy constructs based on transcriptional targeting of genes to cancer cells is a new and promising modality for treatment of cancer. Introducing truncated Bid (tBid), a recently known member of the Bcl-2 family, eradicates cancer cells efficiently. For transcriptional targeting of tBid, two dual-specificity promoters, combining cancer specific core promoters and response modules, were designed. These two core promoter modules contained cancer specific promoters of MUC1 and Survivin genes accompanied by hypoxia-responsive elements and estrogen responsive elements (microenvironment condition of breast cancer cells) which were employed to achieve a higher and more specific level of tBid expression in breast cancer cells. Correlation of the level of tBid expression in normal and cancer cell lines with promoter activity was measured by RT-PCR after treatment with hypoxia and estrogen. The level of tBid expression under control of new hybrid promoters was compared with its expression under control of cytomegalovirus (CMV) promoter as a control. Our data revealed that the level of tBid expression in breast cancer cells were nearly 11 times more than normal cells because of the cancer specific promoters, although tBid expression under control of CMV promoter was almost the same in normal and cancer cell lines. Increased apoptosis was detected in the transfected breast cancer cell lines by the Caspase-3 activity assay. The application of these promoters may prove to have the advantage of tumor selective gene therapy in breast cancer cells and low-potential toxicity for normal tissues.

289. Non-Viral siRNA Delivery for P-gp Downregulation in Breast Cancer Cell Therapy

289. Non-Viral siRNA Delivery for P-gp Downregulation in Breast Cancer Cell Therapy

In vitro cancer cell imaging and therapy using transferrin-conjugated gold nanoparticles

Gold nanoparticles were conjugated with transferrin molecules for targeting, imaging and therapy of breast cancer cells (Hs578T, ATCC). Results show that, the transferrin–transferrin receptor-mediated cellular uptake of gold nanoparticles is six times of that in the absence of this interaction. As a consequence, the laser power effective for photothermal therapy of the cancer cells was reduced to values of two orders of magnitude lower. To demonstrate the efficiency of the conjugated gold nanoparticles in selectively targeting cancer cells, the cellular uptake of the gold nanoparticles by noncancerous cells (3T3, ATCC) was also investigated. The cellular uptake by the normal cells is only one fourth of that by the cancerous cells indicating that the transferrin–transferrin receptor interaction plays an important role in controlling the cellular uptake of the gold nanoparticles.

Integrated molecular targeting of IGF1R and HER2 surface receptors and destruction ofbreast cancer cells using single wall carbon nanotubes

Molecular targeting and photodynamic therapy have shown great potential for selectivecancer therapy. We hypothesized that monoclonal antibodies that are specificto the IGF1 receptor and HER2 cell surface antigens could be bound to singlewall carbon nanotubes (SWCNT) in order to concentrate SWCNT on breastcancer cells for specific near-infrared phototherapy. SWCNT functionalized withHER2 and IGF1R specific antibodies showed selective attachment to breast cancercells compared to SWCNT functionalized with non-specific antibodies. Afterthe complexes were attached to specific cancer cells, SWCNT were excited by∼808 nm infraredphotons at ∼800 mW cm−2 for 3 min. Viability after phototherapy was determined by Trypan blue exclusion. Cellsincubated with SWCNT/non-specific antibody hybrids were still alive after photo-thermaltreatment due to the lack of SWNT binding to the cell membrane. All cancerous cellstreated with IGF1R and HER2 specific antibody/SWCNT hybrids and receivinginfrared photons showed cell death after the laser excitation. Quantitative analysisdemonstrated that all the cells treated with SWCNT/IGF1R and HER2 specificantibody complex were completely destroyed, while more than 80% of the cells withSWCNT/non-specific antibody hybrids remained alive. Following multi-componenttargeting of IGF1R and HER2 surface receptors, integrated photo-thermal therapy inbreast cancer cells led to the complete destruction of cancer cells. FunctionalizingSWCNT with antibodies in combination with their intrinsic optical properties cantherefore lead to a new class of molecular delivery and cancer therapeutic systems.