Background: Breast cancer remains the leading cause of cancer-related deaths among women globally. Despite therapeutic advances, drug resistance and toxicity necessitate discovery of novel bioactive compounds from underexplored natural sources. Insects, particularly cockroaches, produce hemolymph rich in antimicrobial peptides with potential anticancer properties. Blaptica dubia (Dubia roach) is widely cultured as feeder insect but its hemolymph has never been evaluated for anticancer activity. 

Objective: To investigate the in vitro anticancer activity of Blaptica dubia hemolymph against MCF-7 human breast adenocarcinoma cells and assess its selectivity toward normal cells. 

Methods: Hemolymph was collected from adult B. dubia (n=50) by cold immobilization and leg amputation. Cell-free hemolymph was obtained by centrifugation (4,000×g, 15 min, 4°C), filter-sterilized, and protein concentration quantified (Bradford assay). MCF-7 cells and normal human dermal fibroblasts (NHDF) were treated with hemolymph at 25–400 µg/mL for 24, 48, and 72 hours. Cytotoxicity was assessed by MTT assay. Apoptosis was evaluated using acridine orange/ethidium bromide (AO/EB) dual staining and caspase-3 colorimetric assay. Cell cycle distribution was analyzed by propidium iodide flow cytometry. 

Results: B. dubia hemolymph exhibited significant dose- and time-dependent cytotoxicity against MCF-7 cells. The 48-hour IC50 was 112.4 µg/mL. At 400 µg/mL, viability decreased to 21.4% (MCF-7) versus 72.3% (NHDF), yielding a selectivity index of 4.25. AO/EB staining revealed typical apoptotic morphology: chromatin condensation, nuclear fragmentation, and apoptotic bodies (apoptotic index 54.7% vs. 4.2% control). Caspase-3 activity increased 4.2-fold at 200 µg/mL (p<0.001). Flow cytometry showed G0/G1 arrest (68.2% vs. 48.5% control) and increased sub-G1 fraction (22.4% vs. 2.3%). 

Conclusion: Blaptica dubia hemolymph selectively induces caspase-dependent apoptosis and G0/G1 cell cycle arrest in MCF-7 cells. This is the first report of anticancer activity from B. dubia, establishing it as a promising source for bioprospecting novel anticancer lead compounds. Further fractionation and in vivo studies are warranted.