Cytotoxic and Apoptotic Potential of Scylla serrata (Mud Crab) Shell Extract Against Human Cervical Cancer Cell Lines: A Quest for Novel Anticancer Agents

Abstract

Background: Cervical cancer remains the fourth most common malignancy among women worldwide, with
particularly high mortality rates in low- and middle-income countries due to limited access to screening and
treatment. Current chemotherapeutic agents, primarily platinum-based drugs, are associated with severe dose-
limiting toxicities and emerging drug resistance, necessitating the discovery of novel, selective anticancer agents
from natural sources.
Objective: This study aimed to investigate the cytotoxic and apoptotic potential of Scylla serrata (mud crab) shell
extract against human cervical cancer cell lines and elucidate the underlying molecular mechanisms.
Methods: Sequential extraction of S. serrata shells was performed using n-hexane, ethyl acetate, ethanol, and
aqueous solvents. The ethanol extract, which exhibited the most diverse phytochemical profile, was evaluated for
cytotoxic activity against HeLa (HPV-18 positive) and Si Ha (HPV-16 positive) cervical cancer cells and normal
HCerEpiC cells using MTT assay. Apoptosis was assessed through phase contrast and fluorescence microscopy
(Hoechst 33342/PI staining), flow cytometry (Annexin V-FITC/PI and cell cycle analysis), and JC-1 staining for
mitochondrial membrane potential (Δm). Molecular mechanisms were investigated using q RT-PCR for apoptosis-
related gene expression, Western blotting for protein expression, and colorimetric assays for caspase activity.
Results: The ethanol extract demonstrated the most potent cytotoxic activity with IC₅₀ values of 41.3 ± 2.2 μg/mL
(HeLa) and 48.9 ± 2.6 μg/mL (Si Ha) at 72 hours, exhibiting remarkable selectivity indices of 7.23 and 6.10,
respectively, compared to cisplatin (SI < 1.0). The extract induced characteristic apoptotic morphology including
cell shrinkage, membrane blebbing, and nuclear fragmentation. Flow cytometry revealed concentration- and time-
dependent increases in apoptotic populations (up to 58.2% total apoptosis at 2× IC₅₀, 48 hours) with minimal
necrosis (<8%). Cell cycle analysis demonstrated initial G0/G1 arrest followed by pronounced G2/M accumulation
and increased Sub-G1 population. Mechanistically, the extract induced significant dissipation of Δm (70%
reduction in red/green ratio), upregulated pro-apoptotic genes including p53 (4.37-fold), Bax (5.68-fold), Bad
(4.12-fold), caspase-3 (4.85-fold), and caspase-9 (5.24-fold), while downregulating anti-apoptotic Bcl-2 (59%)
and Bcl-xL (47%). Caspase-9 activity showed the greatest increase (up to 7.92-fold), confirming intrinsic pathway
activation, with caspase-3 activation (6.87-fold) and PARP cleavage as downstream execution events.
Conclusion: Scylla serrata shell ethanol extract exhibits potent, selective, and mechanism-based cytotoxicity
against cervical cancer cells through mitochondria-mediated apoptosis involving p53 activation, Bcl-2 family
modulation, and caspase cascade activation. These findings establish S. serrata as a promising, sustainable source
of novel anticancer agents derived from marine byproducts, warranting further bioassay-guided fractionation and
in vivo evaluation.