Abstract P6-12-03: Manumycin A derived nanoparticle induced cytoplasmic vacuolation mediated cell death in triple negative breast cancer

Abstract

Abstract Background: Major treatment modalities of breast cancer therapy are endocrine, radiation and chemotherapies, and can inflict apoptosis resistance effects in most clinically aggressive breast tumors. Among these tumors, triple negative breast cancers (TNBC) are the major aggressive and treatment resistant subtypes. Therefore, it is important to find alternative modes of treatment to eradicate these TNBC and reduce tumor burden. Earlier we have reported the novel role of Manumycin A (ManA), a natural antibiotic produced by Streptomyces parvulus, induced cytoplasmic vacuolation mediated cell death in TNBC. In the present study, nanoparticle based approach was used for delivering ManA to the tumor site to increase the efficacy and reduction of the drug dose. Methods: All cell lines were cultured according to the recommended procedures. Lecithin based nanoparticles were made for both vehicle and ManA. Cell proliferation, transmission electron microscope (TEM), immunoblotting, tumor xenografts and lung metastasis study were performed by standard methods. Results: TEM imaging revealed spherical shaped homogenous size distribution of nanoparticles entrapped with vehicle (Veh-NP) or ManA (ManA-NP). To determine the effect of ManA-NP induced cytoplasmic vacuolation mediated cell death in TNBC, results of ManA-NP were compared to Veh-NP and ManA drug alone. Two fold reduction in the dose of ManA in ManA-NP induced cytoplasmic vacuolation death in several TNBC were observed compared to Veh-NP and ManA drug groups. Indeed ManA-NP caused significantly higher cell death than other groups. ManA-NP induced cytoplasmic vacuolation death was also associated with increased endoplasmic reticulum (ER) stress markers, LC3, p62 proteins and accumulation of ubiquitinated proteins similar to that of ManA alone. Importantly, ManA-NP reduced pAkt and increased PTEN and p21 proteins in TNBC. Notably, apoptotic as well as autophagic inhibitors were unable to protect TNBC against ManA-NP induced cell death. Importantly, thiol antioxidant N-alpha-acetyl-L-cysteine inhibited the formation of cytoplasmic vacuolation mediated cell death along with the induction of ER stress, LC3, p62 and protein ubiquitination in TNBC. Interestingly, ManA-NP failed to induce cytoplasmic vacuolation mediated cell death in slowly dividing normal human mammary epithelial cells even after treating for 72 h at same concentration that is required for ManA drug alone to induce cytoplasmic vacuolation mediated cell death in rapidly proliferating TNBC. Most importantly, ManA-NP reduced breast tumor growth derived from MDA-MB-231 cells in mice at 1 mg/ kg of body weight compared to 5 mg/ kg of body weight of ManA and Veh-NP groups. Finally, ManA-NP completely impeded the lung metastasis of MDA-MB-231 cells compared to Veh-NP mice. Moreover, lung sections of ManA-NP treated mice showed normal thin-walled alveoli structure compared to Veh-NP treated lung sections where cancer cells invaded the lung tissue. Conclusions: These results clearly indicate that ManA-NP enhances the therapeutic efficacy and induces cytoplasmic vacuolation which serves as a "Trojan-Horse" like mechanism of cell death in TNBC. Citation Format: Prajjal K Singha, Srilakshmi Pandeswara, Manjeri A Venkatachalam, Pothana Saikumar. Manumycin A derived nanoparticle induced cytoplasmic vacuolation mediated cell death in triple negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-12-03.