| Summary: | Betulinic acid (3P-hydroxy-Iup-20(29)-ene-28-oic acid) is a pentacyclic triterpene. The
compound can be chemically synthesized from betulin. Both natural compound can be
isolated from the bark of Melalueca cajuputi of a local plant. Betulinic acid (BA) was
reported to possess several biological activities such as anti-inflammatory, anti-viral,
anti-tumor and anti-plasmodial. Selective cytotoxic activity against neuroectodermal
origin cancer and leukemic cancer cell lines were also rep rted, Furthermore, the
compound has been shown to cause apoptosis in a number of cancer cell lines but en
a little to no toxicity against normal cells. The objective of the study was to evaluate
cytotoxic effects and apoptosis cell death of BA on BALB/C murine myelomonocytic
leukemia (WEHI-3B). The cell line was selected because it can be used to induce
leukemia by injection into BALB/c mice which later can be used to evaluate in vivo
anti-leukemic activity. The cytotoxic and anti-proliferative studies ofBA in WEHI-3B
were carried out by using 3-[ 4,5-dimethylthizol-2-yIJ-2,5-diphenyltetrazolium bromide
(MTT) assay. The cytotoxic dose fifty percent (CDso) value was obtained from the dose
response curve of percentage viability against concentration of BA. The anti
proliferative activity was determined by comparing the proliferation of different
cytotoxic doses and also comparing with untreated (control) for 24, 48 and 72 hours
treatment. The effect of compound on cell cycle phases of WEHI-3B cells were
observed by comparing cell cycles profiles of the treated and untreated WEHI-3B cells
analyzed using flow cytometer analysis. Apoptotic and necrotic cells were scored by
observing the cells under the inverted flourescence microscope post staining with
Acridine Orange and Propidium Iodide (AOPI) dyes. Annexin V IPI double staining
was used to recognize stages and characteristics of apoptosis. The mechanism of
apoptosis was studied from the involvement of caspases in caspase activity assay. BA
displayed active cytotoxic activity against BALB/c murine myelomonocytic leukemia
(WEHI-3B), human myeloid leukemia (HL-60) and human breast cancer cell (MCF-7)
but not on human normal colon cells (CCD-18Co), suggesting its action is specific for
tumor cells. MTT assay result showed that the CDsovalue ofBA was 4.471lg/mL and
considered to fall within the range of toxicity effect by the commercial drug,
Vincristine sulphate (VCR). BA was able to show the same inhibitory effect as VCR
on the growth and proliferation of WEHI-3B proved by the optical density of BA
treatment CD dose 25, 50 and 75 were lower than the optical density of control. BA
was found to arrest WEHI-3B at 00/01 phase in a time-dependent manner with
significant result compared to control at 6 hours point (p=0.0001). Vincristine was
found to arrest 02/M phase after 3 and 6 hour followed by 00/01 phase arrest at 12
and 24 hour of treatment with significant cell population percentage reduction
compared to control at 12 (p=0.0024) and 24 hours (p<0.0001). Characteristic and
stages of apoptosis in cells with BA treatment were detected under AOPI and Annexin
V!PI double staining. Membrane blebbing, cell shrinkage and nuclear chromatin
condensation were observed. Apoptotic effects ofBA were very significant (p<0.0001)
when compared to control with throughout the 24,48 and 72 hours. Similarly, apoptotic
percentage of BA treatment also showed very significant result (p<0.0001) when
compared to VCR treatment. The primary mode of cell death in BA treatment was
apoptosis even though there were necrosis detected. Apoptotic cell death percentage
was higher than necrotic cell death when treated with BA for 24,48 and 72 hours. BA
treated cells exhibited early apoptosis showing gradual increase pattern in time
dependent manner. Caspase-8, -9 and -3 activity is also activated in a time-dependent
manner compared to control. BA induced apoptotic cell death in WEHI-3B showing
good potential as an anti-leukemic compound which later can be used in vivo model
studies.
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