inhibition using the MTT assay. Paclitaxel, a standard
commercial drug for treating different cancers, was utilized
as a positive control. The MTT assay is a colorimetric
method based on the metabolic capability of cells to
decrease the yellow tetrazolium salt 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide or MTT to a
blue crystalline formazan product. This method is widely
used to analyze the cytotoxicity and cell viability of living
cells in a 96-well plate.
Figure 1A illustrates the cytotoxicity potential of L.
plantarum 17C and 13C supernatant on HT-29 cancer
cell. Lactobacillus plantarum ATCC 8014 was used as a
reference strain for comparison. The viability of HT-29 carcinoma
cells was inhibited by L. plantarum 17C secretions
after 24 h of incubation, and the viable cells of HT-29 comprised
13% after 24 h of incubation. Our findings revealed
that the antiproliferative effect of L. plantarum 17C secretion
metabolites on HT-29 cancer cells had significant differences
with that of the L. plantarum 13C and L. plantarum
ATCC 8014 as control. We also used normal FHs 74 to
determine the effect of this strain on normal cells (Fig. 1B).
No significant cytotoxic effects for these cells were observed,
and approximately 94% of the normal cells grew well.
Apoptosis detection by DAPI staining
Apoptosis is the primary means of programmed cell death
and serves a significant function in regulating tissue
development and homeostasis. Hence, the induction of
apoptotic cell death is a favorable emerging scheme for
the inhibition and treatment of cancer. Morphological
changes offer the most direct criteria for distinguishing
the apoptotic process. Thus, fluorescence microscopy was
used to detect apoptosis on the basis of changes in
cellular morphology, condensation and fragmentation of
nuclei, as well as cell shrinkage and membrane blebbing.
To scrutinize the effect of L. plantarum 17C on HT-29
cell viability, HT-29 cells were exposed to the supernatant
of the late stationary phase growth of L. plantarum 17C
and analyzed by fluorescent microscopy (Olympus BX61).
None of apoptotic-related signals were observed in the
nontreated HT-29 cells (Fig. 2A). Significant numbers of
apoptotic cells were found in the cells after 24 h of incubation
with 50 lg/mL of L. plantarum 17C supernatant.
The number of apoptotic cells with condensed and fragmented
nuclei was significantly higher than that of blue
intact normal cells. The treated HT-29 cells showed distinctive
signs of apoptosis, including micronucleus formation,
membrane blebbing, cell shrinkage, apoptotic
bodies, and nuclear fragmentation, after 24 h of exposure
(Fig. 2B). Membrane blebbing and micronucleus formation
were prominent apoptotic features of the HT-29 cells
treated with L. plantarum 17C.