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Discussion

Since current therapeutic agents for
chemotherapy of many cancers are not completely effective, and still there are drastic
challenges in this way such as chemoresistance of cancer cells and unwanted
side effects, scientists make effort on finding novel anti-cancer compounds to
decrease these problems as well as possible 1. In recent decades natural compounds derived from medicinal plants
attract much of attention 2-4. Curcumin
is the active ingredient of turmeric that its pharmaceutical properties have
been known from a long time ago 5, 6. Anti-cancer effect is among the most important pharmaceutical
properties of this ingredient that has been approved in several studies in
vitro and in vivo but the molecular mechanism of its action is not clear yet 7-10. On
the other hand, low stability and bioavailability of curcumin limit its
clinical application 11. So nowadays several curcumin nano-structures have been developed
that overcome these barriers 12. Dendrosomal nano-curcumin (DNC) is one of those which improve the
solubility, cellular uptake and toxicity of curcumin 13, 14. Based on mentioned points in this study we aimed to apply curcumin
as a toxic agent for breast cancer cells. This is the first study to evaluate
the anti-cancer ability of DNC on T47D with p53 mutation, a gene that its
mutated form observed in over 50% of human cancers.

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In the first step, we performed MTT
assay to test the cytotoxicity of DNC and our data showed that DNC
significantly affects cellular viability of T47D/MCF-7 cells in a time- and
dose-dependent manner; whereas, pristine curcumin as well as pristine
dendrosome did not show any remarkable toxicity at the same concentrations as
the nano-based compound (data for curcumin and dendrosome not shown).
Therefore, only DNC was investigated in further assessments. Meanwhile, we
previously reported that DNC does not affect mouse normal fibroblasts (MEF) and
human peripheral blood mononuclear cells (PBMC) 14. Furthermore to detect the mode of induced cell death we applied
flowcytometry test using Annexin V/FITC and western blot analysis for PARP
cleavage assays. As descried in results section, DNC has induced apoptosis in
T47D cells in a time-dependent manner and no necrosis detected following
treatment, which is in consistence with previous reports. Similar
results were achieved for MCF-7 cells. Effect of DNC on suppressing cancer cell
proliferation via induction of apoptosis has been reported earlier in different
cell lines including AGS (gastric) 15, WEHI-164 (fibrosarcoma) 12, A431 (epithelial) 12, U87MG (Glioma) 16, SW480 (colon) 17 and HepG2/Huh7 (Hepatocarcinoma) 18.

In the next step, to better discernment
of the molecular mechanism of DNC mediated apoptosis, the mRNA expression level
of BAX, Bcl-2, survivin and p53 witch are important regulators of apoptosis
were measured in the DNC-treated T47D and MCF-7 cells by real-time PCR
analysis. Our results indicated that DNC significantly decreases mRNA
expression of the anti-apoptotic Bcl-2 and survivin genes. However, p53
expression has been decreased in both wilde-type and mutant forms. Bax and Bcl2
are the main members of Bcl-2 family and regulate mitochondrial apoptosis
pathway and previous reports indicated the effect of curcumin on increasing the
expression ratio of BAX/Bcl-2 genes in HCT116, HT29 18, 19 and
AGS 15 and MDA-MB-231 cells 20. Moreover survivin is an anti-apoptotic gene that inhibits cell
death by suppressing the function of caspase 3 and 7 21 and its under-expression has been shown in several studies. Glienke
et.al indicated the curcumin impact on down-regulation of survivin gene
expression at both the transcriptional and translational level of pancreatic
cancer cell lines 22.

On the other hand, to partially
uncover the reason for the high cellular accumulation of DNC versus pristine
curcumin, efflux and cell swelling assays were performed to see whether
curcumin modulates the function of p-glycoprotein, as a well-known active
transporter that conveys cytosolic compounds out of cells. Several researches
have shown that dysfunction of the p-gp membrane pump creates cell swelling,
addressing its role in cell volume-regulated chloride conductance path 23, 24. Our data represented that fluorescent Rho123 is significantly
accumulated in DNC-treated T47D and MCF-7 cancerous cells as well as verapamil
(positive control)-treated cells. Enhancement of intracellular Rh123 is related
to the capacity of DNC to bind the P-gp trans-membrane pump. Also results from
cell swelling assay illustrated that treating cells with DNC impairs cellular
trans-membrane pumping protein P-gp which plays a major role in controlling
cellular osmotic pressure when cells are exposed to hypotonic medium. In many
studies, resistance to the cytotoxic drugs has most often been linked to the
overexpression of P-gp and dysfunction of this membrane pumping protein
probably can reduce cellular resistance to chemotherapy strategies 25-27.
However, this is the first report demonstrating the suppressive effect of DNC
on the activity of the multidrug resistance p-glycoprotein, which may lead to
the accumulation of compounds inside the cytosol.  

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