Abstract Cupredoxins are a family of low-molecular weight, water soluble, copper-containing redox proteins involved mainly in the electron transfer chain in prokaryotes. Azurin is a member of the cupredoxin family secreted by Pseudomonas aeruginosa, of which a 28-amino acid sequence (p28) was identified to acquire the capability of preferential entry into cancer cells of various histogenesis. Upon entry, it exerts a cytostatic effect in these cancer cells via a p53-mediated pathway. Our aim was to study whether the other members of the cupredoxin family possess a similar function to that of azurin in the eukaryotic cancer cells. Based on structural and sequence analyses, helical regions of auracyanin A & B were shown to resemble p28 closely. Thus, we studied the biologic function of two 28-amino-acid peptides derived from auracyanin A (aurA; pI 4.5, MW 2.6 kD), and auracyanin B (aurB; pI 3.4, MW 2.7 kD). Cancer growth and MTT assays demonstrated that aurB, but not aurA, induced cytotoxicity in various cancer cell lines in a dose-dependent (range 0.1uM-100uM) and time-dependent (~72 hours) pattern, including p53-null (prostate cancer PC-3) and dn-p53 (breast cancer MDD-2 and ovarian cancer SKOV-3) in which p28 did not demonstrate a significant growth inhibition. Annexin-V assays showed a dose-dependent apoptosis in prostate cancer cell lines of various p53 statuses (wt-p53 LNCaP, mut-p53 DU-145, p53-null PC-3) following treatment with aurB at 5-50uM for 48 hours, whereas p28 did not demonstrate an apoptotic activity in p53-null cell lines, suggesting an aurB-induced apoptosis via a p53-indepdnent pathway. In contrast, aurA did not appear to induce apoptosis in any of the above cell lines. Induction of apoptosis was mediated through depolarization of the mitochondrial membrane and caspase cascade activation in cancer cells. Moreover, fluorescence microscopy demonstrated that aurB, unlike p28, does not acquire a similar pattern of preferential entry into cancer cells. This proposes that aurB acquires the potential as an anticancer cell penetrating peptide, and differs from p28 in its mode of entry and p53-mediated action. Our studies pertaining to azurin and auracyanin, suggest that cupredoxins, as a family of prokaryotic metalloproteins, could represent a fountainhead of numerous derived peptides that hold promise as novel and efficient antineoplastic agents against various cancer cell lines. Citation Format: Samer A. Naffouje, Tohru Yamada, Tapas K. Das Gupta. A peptide derived from a photosynthetic cupredoxin protein, auracyanin, induces a p53-independent apoptosis in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2311. doi:10.1158/1538-7445.AM2017-2311