الفهرس 
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Abstract
Cancer has become the leading cause of death worldwide and seriously endangering
the health and life of humans. Breast cancer is a very common malignant tumor among female
patients and it is estimated that 1 in 10 women worldwide is affected by breast cancer during
their lifetime. However, chemo- and radiotherapy are still the most common treatment options
for breast cancer; resistance invariably becomes a major issue. A wide variety of mechanisms
have been implicated in drug resistance of cancer cells including increased or decreased
expression of proteins, reduced apoptosis via alteration in the expression of Bcl-2 family
members. Also, the non-specific action of chemotherapeutics is associated with many side
effects. Chemotherapeutics affect non-selectively target actively proliferating cells that lead to
the destruction of healthy and cancerous cells. Chemotherapy can be toxic to the patient with
a number of side effects and risks. Thus, it is very important to search for new therapeutic
agents with less resistance potential to treat breast cancer arresting mitotically active cells,
reducing cancer stem cell populations, and inducing apoptosis. For over 50 years, natural
products have served well in combating cancer. Recently, apoptosis has been reported to be
the cause of tumor cell death during chemotherapy and radiation therapy.
The main aim of the present study was to investigate the anticancer activity of
violacein, phycocyanin, phycocyanobilin, and both violacein-phycocyanin in combination
through studying their effect on inducing apoptosis and glycan profiling.
This study was an in vitro study employing the human breast cancer cell line MCF-7
that divided into 6 groups as following:
group I : MCF-7 cell line maintained in drug free environment as untreated control.
group II : MCF-7 cell line treated with serial concentration of etoposide as
conventional anticancer chemotherapeutic agent
group III : MCF-7 cell line treated with serial concentration of violacein
group IV : MCF-7 cell line treated with serial concentration of phycocyanin
group V : MCF-7 cell line treated with serial concentration of phycocyanobilin
group VI : MCF-7 cell line treated with serial concentration of violacein and
phycocyanin in combination.
Human breast cancer cell line MCF-7 metabolic activity and viability (reflecting their
survival and growth) was determined for all groups with MTT assay. The relative expression
of Bax, Bcl2, caspase 3 was determined for all groups at IC50 by real-time reverse
transcription polymerase chain reaction (RT-PCR). Also, the variation in expression of Nglycan
and O-glycan was determined using MALDI-TOF.
The present study revealed that all studied compounds decrease the cell viability (%)
of MCF-7 cells in a concentration dependent manner. The antiproliferative effect of violacein
may be achieved by its capability for entailing production of reactive oxygen species (ROS)
in cancer cell, which results in the triggering of apoptosis. Phycocyanin could effectively
inhibit the proliferation, induce tumor G0/G1 cell cycle arrest and promote the apoptosis. The
antiproliferative effect of phycocyanobilin was partially attributed to their potent antioxidant
activity.
The combination of two or more therapeutic treatments to specifically target cancerinducing
or cell sustaining pathways is a cornerstone of cancer therapy.The obtained results
revealed that the combination of violacein and phycocyanin generated synergistic anticancer
effect in MCF-7 cells (CI<1) at violacein concentration less than 1.25 μg/ml.
In the present study, the relative gene expression results indicated the all studied
compounds up-regulated the proapototic gene (Bax) and the caspase-3 gene expression. The
Bcl2 expression was down-regulated in the etoposide and combined therapy treated cells only.
However, there was up-regulation of Bcl2 expression in violacein, phycocyanin, and
phycocyanobilin treated cells. The upregulation of Bcl2 expression may be attributed to the
molecular machinery effort of the cell to survive and escape apoptotic cell death. The
alteration in both Bax and Bcl2 expression lead to altering the Bax/Bcl2 ratio which
significantly increased in etoposide, violacein, and combined therapy treated MCF-7 cells as
compared with untreated cells which results in activation of caspase3 and release of
cytochrome C.
Moreover, the results of morphological analysis revealed that the studied compounds
produced morphological alterations which were typical of the apoptotic process such as
shrinkage, membrane abnormalities with membrane blebbing, appearance of cytoplasmic
vacuoles, appearance of apoptotic bodies and disappearance of microvilli.
Regarding the effect of the studied compounds on the expression of N-glycan and Oglycan,
the present study revealed that the violacein, and combined treatment had a significant
reduction in the formation of tri- and tetra-sialylated N-glycan structure and di-sialylated Oglycan
structure. The most potent one was the combined therapy (violacein-phycocyanin).
Since, hypersialylation of the Fas receptor was shown to disable apoptosis induction in cancer
cells, the reduction in the expression of sialylated glycan enabled the induction of apoptosis
through extrinsic pathway. Furthermore, there was no effect on the intensity of some types of
high mannose structure with single or combined treatment with violacein or phycocyanin
while there were an elevation in the intensity of others high mannose structures (structure 5
and 7). The elevation in intensities of high mannose glycans suggests a premature termination
of the glycosylation pathway and a problem during the synthesis that prohibits the deletion
and subsequent addition of sugar residues. Indeed, mechanistic deviations in the earlier part of
a biosynthetic pathway are known to limit the amounts of specific structural types and cause
an increase in the amount of other structures. The increased presence of high mannose glycans
has consequences for the function of the protein.
The altered expreesion of N- and O-glycans in MCF-7 cell line treated with violacein
and/or phycocyanin indicated that glycans serve an important role in tumor growth and
progression. So glycans can be used as a target for cancer therapy.