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Apoptosis inducing capacity of Holothuria arenicola in CT26 colon carcinoma cells in vitro and in vivo.
Baharara J
,
Amini E
,
Afzali M
,
Nikdel N
,
Mostafapour A
,
Kerachian MA
.
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OBJECTIVES: Sea cucumber is one of the classes of echinoderms, which is considered as a health marine product and possess various biological characteristics with therapeutic application. The present investigation attempted to evaluate the potential of anti-cancer Persian Gulf sea cucumber species Holothuria arenicola (H. arenicola) aqueous extract on mice colon carcinoma cells in vitro and in vivo.
MATERIALS AND METHODS: The CT26 carcinoma cells were treated with various concentrations of extract in 24 and 48 hr, and then its anti-proliferative effect was measured by MTT assay and morphological observations. The apoptotic effect was examined by fluorescence microscopy (DNA fragmentation assay), Flow cytometry, caspase-3 and -9 colorimetric assays. The in vivo anti-tumor efficacy of sea cucumber extract on CT26 tumor cells transplanted in BALB/c mice was also investigated.
RESULTS: The results showed that the water extract of sea cucumber revealed remarkable anti-proliferative effect on CT26 tumor cells with IC50= 31 µg/ml with recruitment of intrinsic apoptotic pathway in vitro. In addition, the colon tumor volume in treated groups remarkably reduced in homozygous mice. Histopathological examination elucidated that sea cucumber extract attenuated tumor size and volume along with apoptosis characteristics. Moreover, RT-PCR analysis revealed that sea cucumber extract induced intrinsic apoptosis in vivo through suppression of Bcl-2 expression.
CONCLUSION: Our data confirmed this notion that sea cucumber administrates anti-cancer effect that can be used as complementary in preclinical experiments, so further characterization are recommended for detection sea cucumber metabolites and clinical application.
Figure 2. Morphological evaluation of CT26 colon cancer cells treated with sea cucumber extract. A) left to right; untreated cells, CT26 cells treated with 20, 40, 100 µg/ml extract for 48 hr, respectively. B) The same classification after MTT assay, as shown in Figure 2B, formazan crystals are abundant in control group and duration treatment with enhancement dosage of sea cucumber extract, decreased formazan crystals
Figure 3. a) Fluorescence images of AO /PI stained CT26 colon cancer cells treated with sea cucumber extract. A) Untreated cells with intact nuclei (green color) B) treatment with 20 µg/ml, C) treatment with 40 µg/ml and D) treatment with 100 µg/ml sea cucumber extract. As shown, yellow, orange color is signs of apoptosis. b) DAPI staining exhibited chromatin fragmentation and nuclear disintegration. A) Untreated cells with normal nuclei, B) 20 µg/ml, C) 40 µg/ml and D) 100 µg/ml treatment with sea cucumber extract
Figure 4. Flow cytometry analysis of untreated CT26 cells and with various concentrations of sea cucumber extract. A) CT26 cells without treatment, B) CT26 cells under treatment with 20µg/ml, C) CT26 cells exposed with 40µg/ml sea cucumber extract as IC50 value
Figure 5. Effect of sea cucumber extract on caspase-3 and -9 activity in CT26 cells. Histogram represented caspase-3 and -9 activity in untreated cells and with 40, 60µg/ml sea cucumber extract
Figure 6. A) CT26 colon carcinoma cell line, mouse bearing colon cancer, histological section from tumor inoculated, (left to right, respectively). B) The alterations in tumor volumes in control and experimental groups during 14 days exposure with sea cucumber extract. All data are provided as mean ± SD. Sea cucumber extract suppress CT26 derived tumor volume. C) Evaluation of body weight in tumor-bearing mice treated with sea cucumber extract. There are no significant differences in body weight between experimental and control groups
Figure 7. Effect of aqueous sea cucumber extract on CT26 tumor bearing mice. a) Morphological changes in tumor size A) control group and B, C, D) under treatment with sea cucumber extract (50, 100, 400 mg/kg body weight). b) Light microscopic observations on tumor structure (400×) of tumor cross sections in control group (A) and 100, 400 mg/kg experimental groups (B, C) respectively, indicating pyknotic nuclei in experimental group C
Figure 8. Effect of sea cucumber extract on expression of Bcl-2 mRNA in colon tumor cells. B2M mRNA was used as an internal control gene (230bp). Lane left to right: 50 bp molecular weight marker, control, 100 and 400 mg/kg extract, respectively. As shown in the figure, down regulation of Bcl-2 under treatment sea cucumber extract elucidated intrinsic apoptotic induction on colon tumor cells
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