Human SP-D Acts as an Innate Immune Surveillance Molecule Against Androgen-Responsive and Androgen-Resistant Prostate Cancer Cells

ORIGINAL RESEARCH published: 11 July 2019 doi: 10.3389/fonc.2019.00565 Human SP-D Acts as an Innate Immune Surveillance Molecule Against Androgen-Responsive and Androgen-Resistant Prostate Cancer Cells Gargi Thakur 1 , Gagan Prakash 2 , Vedang Murthy 2 , Nilesh Sable 2 , Santosh Menon 2 , Salman H. Alrokayan 3 , Haseeb A. Khan 3 , Valarmathy Murugaiah 4 , Ganesh Bakshi 2 , Uday Kishore 4 and Taruna Madan 1* 1 Edited by: Massimiliano Berretta, Centro di Riferimento Oncologico di Aviano (IRCCS), Italy Reviewed by: Gaetano Facchini, National Cancer Institute G. Pascale Foundation (IRCCS), Italy Giuseppe Palma, National Cancer Institute G. Pascale Foundation (IRCCS), Italy *Correspondence: Taruna Madan Specialty section: This article was submitted to Molecular and Cellular Oncology, a section of the journal Frontiers in Oncology Received: 29 March 2019 Accepted: 10 June 2019 Published: 11 July 2019 Citation: Thakur G, Prakash G, Murthy V, Sable N, Menon S, Alrokayan SH, Khan HA, Murugaiah V, Bakshi G, Kishore U and Madan T (2019) Human SP-D Acts as an Innate Immune Surveillance Molecule Against Androgen-Responsive and Androgen-Resistant Prostate Cancer Cells. Front. Oncol. 9:565. doi: 10.3389/fonc.2019.00565 Frontiers in Oncology | www.frontiersin.org Department of Innate Immunity, ICMR-National Institute for Research in Reproductive Health, Mumbai, India, 2 Tata Memorial Hospital, Homi Bhabha National Institute, Mumbai, India, 3 Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia, 4 Biosciences, College of Health and Life Sciences, Brunel University London, Uxbridge, United Kingdom Surfactant Protein D (SP-D), a pattern recognition innate immune molecule, has been implicated in the immune surveillance against cancer. A recent report showed an association of decreased SP-D expression in human prostate adenocarcinoma with an increased Gleason score and severity. In the present study, the SP-D expression was evaluated in primary prostate epithelial cells (PrEC) and prostate cancer cell lines. LNCaP, an androgen dependent prostate cancer cell line, exhibited significantly lower mRNA and protein levels of SP-D than PrEC and the androgen independent cell lines (PC3 and DU145). A recombinant fragment of human SP-D, rfhSP-D, showed a dose and time dependent binding to prostate cancer cells via its carbohydrate recognition domain. This study, for the first time, provides evidence of significant and specific cell death of tumor cells in rfhSP-D treated explants as well as primary tumor cells isolated from tissue biopsies of metatstatic prostate cancer patients. Viability of PrEC was not altered by rfhSP-D. Treated LNCaP (p53+/+ ) and PC3 (p53 −/− ) cells exhibited reduced cell viability in a dose and time dependent manner and were arrested in G2/M and G1/G0 phase of the cell cycle, respectively. rfhSP-D treated LNCaP cells showed a significant upregulation of p53 whereas a significant downregulation of pAkt was observed in both PC3 and LNCaP cell lines. The rfhSP-D-induced apoptosis signaling cascade involved upregulation of Bax:Bcl2 ratio, cytochrome c and cleaved products of caspase 7. The study concludes that rfhSP-D induces apoptosis in prostate tumor explants as well as in androgen dependent and independent prostate cancer cells via p53 and pAkt pathways. Keywords: pattern recognition receptor, prostate tumor explants, LNCaP cells, PC3 cells, viability, apoptosis, p53, pAkt 1 July 2019 | Volume 9 | Article 565 Thakur et al. Anti-prostate Cancer Activity of rfhSP-D INTRODUCTION To take the next logical step from the reported anticancer role of SP-D in tumorigenic cell lines, we examined anti-prostate cancer role of rfhSP-D using tumor explants and primary cells derived from tissue biopsies of metastatic prostate cancer patients. rfhSP-D induced apoptosis selectively in various prostate cancer cells including the two prostate cancer cell lines (LNCaP and PC3) in a dose- and timedependent manner. Apoptotic signaling involved upregulation of p53 and downregulation of pAkt. Decreased levels of Bcl2, with a concomitant increase in Bax, cytochrome c and cleavage of caspase 7, confirmed rfhSP-D mediated induction of intrinsic apoptosis. Prostate cancer, an adenocarcinoma of epithelial cell-origin, is the second most frequently diagnosed cancer among men (1). In its early stages, prostate cancer cells rapidly proliferate in an androgen dependent manner, and thus, are treated by androgen deprivation therapy (2). Conventional anti-cancer treatments such as chemotherapy and radiotherapy improve survival, but many patients encounter relapse and metastasis. Following the remittent stage, the cancer progresses to become androgenindependent where most therapeutic strategies fail. Immunotherapy by stimulating pattern recognition receptors of the innate immune system such as toll-like receptors (TLRs) has shown promise as a preferred adjunct treatment against cancer (3). Imiquimod, a synthetic imidazoquinoline and an agonist that targets TLR-7 and induces the production of proinflammatory cytokines including IFN-α, IL-6, and TNF-α, inhibited cancer growth in the mouse prostate via apoptosis induction (4, 5). Collectins are pattern recognition proteins belonging to the C-type lectin family. They are composed of an N-terminal cysteine-rich region, a triple-helical collagen domain, an αhelical coiled-coil neck region, and C-terminal carbohydrate recognition domain (CRD) (6). Surfactant Protein D (SP-D) is one of the most studied collectins with a vital role in host defense against pathogens and allergens, and modulation of inflammatory response (6). Although SP-D was historically shown to be lung-resident being produced by type II alveolar and Clara cells (7), studies during the last decade have established its extrapulmonary existence in a range of tissues (8). SP-D has also been shown to be expressed in the male reproductive tracts of human and mice (9, 10). Elevated levels of SP-D at inflamed sites in the prostate manifested protection against bacterial infection (11, 12). Kankavi et al. (13) observed differential expression of SP-D in glandular structures of inflamed malignant and non-malignant human prostate tissues. There was a significant correlation between decreased levels of SP-D and increased Gleason score, a grading system based on the histologic pattern of arrangement of carcinoma cells, and tumor volume. Previously, we reported a novel anti-cancer role of human SP-D and its recombinant fragment (rfhSP-D) comprising 8 Gly-X-Y repeats neck and CRD region, wherein they reduced the viability of a range of human cancer cell lines including eosinophilic leukemia cell line (AML14.3D10) (14). Importantly, survival of peripheral blood mononuclear cells (PBMCs) derived from healthy individuals was found to be unaltered (14). rfhSPD treated AML14.3D10 cells showed a significant increase in apoptosis with reduced HMGA1 levels and increased le (...truncated)