Clinacanthus Nutans Hexane Extracts Induce Apoptosis Through a Caspase-Dependent Pathway in Human Cancer Cell Lines

Background: Clinacanthus nutans (C.nutans) is a plant consumed as a cancer treatment in tropical Asia. Despite
the availability of numerous anecdotal reports, evaluation of active anticancer effects has remained elusive. Therefore
we here examined antiproliferative, reactive oxygen species (ROS)-inducing and apoptosis mechanisms of whole plant
extracts in different cancer cell lines. Methods: Antiproliferative actions of five solvent extracts (hexane, chloroform,
ethyl acetate, methanol and water) of C.nutans were tested on non-small cell lung cancer (A549), nasopharygeal cancer
(CNE1) and liver cancer (HepG2) cells using MTT assay. The most potent anticancer extract was then assessed by flow
cytometry to study cell cycle changes . Intracellular levels of ROS were quantified by DCFH-DA assay. Involvement of
the caspase pathway in induction of apoptosis was assessed using caspase assay kits. GC-MS analysis was performed
to identify phytoconstituents in the extracts. Results: Hexane and chloroform extracts were antiproliferative against
all three cell lines, while the ethyl acetate extract, at 300 μg/mL, was antiproliferative in the CNE1 but not A549 and
HepG2 cases. Methanol and water extracts did not inhibit cancer cell proliferation. The most potent anticancer hexane
extract was selected for further testing. It induced apoptosis in all three cell lines as shown by an increase in the
percentage of cell in sub-G1 phase. Dose-dependent increase in ROS levels in all three cell lines indicated apoptosis to
be possibly modulated by oxidative stress. At high concentrations (>100 μg/mL), hexane extracts upregulated caspases
8, 9 and 3/7 across all three cell lines. GC-MS analysis of the hexane extract revealed abundance of 31 compounds.
Conclusion : Among the five extracts of C.nutans, that with hexane extract demonstrated the highest antiproliferative
activity against all three cancer cell lines tested. Action appeared to be via ion of intracellular ROS, and induction of
apoptosis via intrinsic and extrinsic caspase pathways.