In Vitro and In Vivo Investigation of Locally Isolated Microalgae Strains for their Anti-Cancer Activity and Protective Effects against Heavy Metal Carcinogenicity

Abstract

Heavy-metal exposure, notably nickel (Ni), contributes to lung carcinogenesis through oxidative stress, hypoxia signaling, and epithelial-mesenchymal transition (EMT). This thesis investigated the anticancer potential of locally isolated microalgae for bioactivities relevant to Ni-carcinogenesis risk mitigation. Porphyridium purpureum (QUCCCM160) was prioritized for its high antioxidant capacity, proven Ni-biosorption, and favorable PE-extract properties (BET/cellular uptake), and was investigated for its anticancer and metallo-protective effects. The anticancer activity was assessed using real-time cell analysis (RTCA) and Cell Counting Kit-8 (CCK-8) assays, revealing significant dose-dependent and selective inhibition of cancer cell proliferation across NSCLC cell lines. The IC50 values of the PE-rich extract on NCI-H1975, A549, and NCI-H322 were 14.87±2.64, 12.75 ± 3.42, and 11.73±1.58 μg/mL, respectively, compared to 41.19 ± 2.51 μg/mL for LL24 normal cells. Morphological observations and wound healing assays showed apoptotic-like changes and reduced migratory ability. Human apoptosis array confirmed the activation of both intrinsic and extrinsic apoptotic pathways. RT-qPCR data showed significant modulation of epithelial-mesenchymal transition (EMT) and migration-related gene expressions in a cell line– and stage–dependent manner, consistent with apoptosis induction and migration restraint. The in vivo efficacy was evaluated in the zebrafish xenograft model, where Dillabeled A549 and NCI-H1975 (primary) and NCI-H322 (metastatic) NSCLC cells were injected into the zebrafish yolk and treated with 10 μg/mL PE-extract. Fluorescence imaging at 24 and 72 hours post-injection revealed that PE treatment significantly inhibited tumor growth in the primary (NCI-H1975, A549) cell xenografts, while NCI-H322 cells showed lower sensitivity, likely due to their metastatic aggressiveness. Finally, metallo-protection was tested by cell exposure to Ni and pre-treatment with PE-extract. RTCA and wound-healing assays demonstrated attenuation of Ni²⁺-induced toxicity in both A549 and LL24, with preferential protection of non-malignant LL24 fibroblasts. In LL24, PE-extract reduced pro-inflammatory transcripts (TNFα, IL-1β, IL-6) and partially restored CDH1, in A549, PE-extract reduced IL-1β and further inhibited hypoxia/EMT drivers (HIF-1α, ZEB1). Collectively, PE-extract combines anticancer activity in NSCLC (in vitro and in vivo) with metallo-protective effects against Ni²⁺-toxicity, showing selective protection of normal cells while constraining tumor-promoting programs. This dual profile supports the development of standardized PE-based interventions for adjunctive cancer prevention and mitigation of environmental Ni exposure impacts.