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Summary
The main objectives of the present study were to determine the activity of
the soluble β-glucan (local and commercial extracts) of Saccharomyces
cerevisiae to induce cytotoxic effects on cancer cells as in vitro and in vivo
studies by using specific parameters . The present study based on the
preparation of solube β-glucan and the determination of their polysaccharides
,the LD50 and liver enzyme alanine amino transferase (ALT) were determined .
In addition, study the non-specific immune response through detection of the
phagocytic activity with cytogenetic study of mice bone marrow ; and using
ELISA reaction to detect the Transforming Growth Factor beta 1(TGFß1) level
in mice ; as well as study the histopathological changes and the terminal Bax
gene and Bcl-2 gene expression by using in situ hybridization technique to
detect the induction of the apoptosis in spleen and tumour tissue sections .
In vitro study the local and commercial extracts showed a concentration
and time dependence growth inhibitory effects of beta glucan. Therapeutic dose
demonstrated a potent cytotoxic effect in mammary adenocarcinoma (AMN-3)
cell line in high concentration (500 ,1000)µg/ml which represented
(92% and 94%),(87%,85%) respectively, after 48hrs of exposures . While the
low concentrations (5,50)µg/ml caused low level in the growth inhibition for
AMN-3 cell line after 24hrs and 72hrs of exposures, except at the 48hrs the
cytotoxic effect of local and commercial extracts represented (89%,89%),(90%
and 91%) respectively , while the concentrations caused growth stimulations
and there viability for Rat embryo fibroblast (Ref) cells . The cytological study
performed simultaneously with cell growth assay, revealed that there was
concentration-dependant cytological changes for AMN-3 like patchy growth
inhibition , loss of confluent feature and cellular degeneration after exposure to
the lowest concentrations (5,50)µg/ml . While the highest concentrations
(500,1000) µg/ml caused sever growth inhibition with marked cytolytic features
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including loss of cellular outline and high content of cellular debris for both
treatments at 48hrs of exposure .
The results of the study showed no LD50 and ALT liver enzyme effect of
the concentrations (200,400,600,800,1000 µg/ml) used in experiment for local
extract of β-glucan during 24hrs(LD50) , 21days (ALT) in mice inoculation. The
therapeutic effects have been detected in tumor-bearing mice by Subcutanous
administration of soluble β-glucan (local and commercial extracts) at a dose of
1mg/ml showed reduction in relative tumor volume (216,241)mm3for groups
that treated after tumor cell line transplantation (G1) and (188, 212)mm3 for
groups that treated after 24hrs of tumor transplantation (G3) when compared to
the control group (478 , 389)mm3 respectively , and tumor growth inhibition
(54.4% , 49.5%) for G1,and (51.6%, 45.5%) for G3 respectively, A relative
tumour volume and tumor growth inhibition did not observed in group G2(that
treated before tumor cell implantation).The cytogenetic study on bone marrow
cells in transplanted mice after treatment with β-glucan (local and commercial
extracts) of S. cerevisiae for all groups showed a significant increase (P<0.05)
in mitotic index (MI) of bone marrow cells and a high significant increase
(P<0.05) by both treatment and in all durations when compared to the control
groups .The phagocytic activity by carbone clearance showed a significant
increase (p<0.05) in G4 groups treated with local extract of β-glucan which
high value (0.08) than commercial (0.02) as compared with the control group
G6 (0.006). The data revealed a significant increase (p<0.05) in the level of
Transforming Growth Factor (TGFβ1) in the group G6 (4.71ng/ml) as
compared with the PBS-treated control G5 group (3.77ng/ml) ,TGF-β1 level
was detected as increased in value from (2.60ng/ml) in G2 to (4.35ng/ml) in
G1,G3 for both treatments .
The results of in situ hybridization analysis (ISH) of tumor and spleen
tissues showed that the β-glucan can accelerate apoptosis in AMN-3 tumor cell
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by up-regulation of Bax (pro-apoptosis) and down-regulation of Bcl-2
(anti-apoptosis) expression. The mean percentage of Bax expression showed
low values in group (G5) (11.37%) than Bcl-2 expression (71.34%), the high
value was determined in (G1 & G2) for both local and commercial extracts
groups were represented (53.46% ,43.93%) for G1 and (52.23%,54.77%), for
G2 respectively . A low Bcl-2:Bax ratio attributed to a high Bax expression
(54.7%) with low Bcl-2(30.07%) was significantly lower (P<0.05)than the
control group , which indicated that the β-glucan had a good therapeutic effect
on tumor cell and the potential of clinical application as a chemotherapeutic
agent . Histopathological study on tumour bearing mice groups that treated with
both treatments by which that the spleen showed a follicular hyperplasia due to
increasing in white pulp , hemorrhage of parenchymal tissue and the presence
of megakaryocytes infiltration . The kidney and liver sections showed an
inflammatory mononuclear cells infiltration and a few changes in the normal
tissue were noticed after treatment by local and Commercial extracts . The
therapeutic β-glucan dose of tumor bearing mice groups showed an area of very
aggressive tumor (solid anaplastic tumor) , the central area appeared wide of
destruction of malignant tissues with necrosis in most of the cancer cells tissue,
homogenous structures and associated with acute inflammatory hemorrhage cell
infiltrated with cellular depress of polymorphnuclear cells and high number of
mitotic figure . The results obtained from this study clearly indicated that
soluble β-glucan have anti-tumor effect in vitro and in vivo, potent phagocytic
carbone clearance effect and apoptotic gene expression.
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