الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
For decades, cancer has been hailed by the somatic mutation theory (SMT), which cites that the initiating disease process is the result of nuclear mutations in oncogenes and tumor suppressor genes. However, the mitochondrion theory, in contrast with the SMT, appears to be the only cancer initiation theory that relates to all cancers. Mitochondria contributes to malignant transformation by at least three major mechanisms: (1) Mitochondrial ROS causes the accumulation of potentially oncogenic DNA defects and the activation of potentially oncogenic signaling pathways. (2) The abnormal accumulation of specific mitochondrial metabolites, including fumarate, succinate, and 2-hydroxyglutarate, has prominent transforming effects. (3) Functional abnormalities in MOMP or MPT are required for the survival of neo-formed malignant precursors., which would otherwise succumb to regulated cell death.
Doxorubicin is one of the most effective and widely used anticancer anthracycline drugs. The anticancer effects of Doxorubicin are believed to occur through the inhibition of topo II enzyme and subsequent blockage of DNA resealing during cell replication. Decreased dosage suggests reduced anticancer efficacy and poor survival. However, curative doses are often related to severe cardiotoxicity, including life-threatening cardiomyopathy and congestive heart failure. Thus, it is very important to search for a new therapeutic agent that can increase the effectiveness of Low doses of Doxorubicin, with least cell toxicity, to have a more effective treatment for breast cancer with least side effects.
Doxycycline is a tetracycline-derivative, wide-spectrum antibiotic, which was first approved by FDA in the late 1960s. Tetracyclines inhibit protein synthesis by interfering with the binding of activated aminoacyl-tRNAs on the A-site of the 30S subunit of bacterial ribosomes.
Given the endosymbiotic origin of mitochondria, the 28S mitochondrial ribosome is homologous to the 30S bacterial ribosome which accounts for the manageable side-effects of Doxycycline. Thus, we propose to treat cancer as an infectious disease, by repurposing FDA-approved antibiotics that inhibit mitochondrial biogenesis as a known side effect, that could be directed instead as a therapeutic effect.
So, the main target of this study was to:
1. Investigate the possible antitumor effect of Doxycycline in comparison to Doxorubicin in human breast cancer cell line MCF-7.
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2. To elucidate the possibility of applying Doxycycline as an adjuvant therapy in Doxorubicin-based regimes in vitro.
3. To test the possible role of Doxycycline on the expression of Bcl-2 and the release of Cytochrome C from the mitochondria
This study was an in vitro study employing the human breast cancer cell line MCF-7 that divided into four groups:
group I : MCF-7 cell line cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) as untreated control. group II : MCF-7 cell line cultured in DMEM containing different concentrations of Doxorubicin (0.01-10 M) as an anti-cancer chemotherapeutic agent. group III : MCF-7 cell line cultured in DMEM containing Doxycycline (25-200 M). group IV : MCF-7 cell line cultured in DMEM containing combination of Doxorubicin and Doxycycline.
The following studies were performed for all groups:
1. Human breast cancer cell line MCF-7 metabolic activity and viability (reflecting their survival and growth) was determined with MTT assay.
2. Total RNA was extracted from MCF-7 cell line and the expression of Bcl-2 was assayed for all groups by one step real-time reverse transcription polymerase chain reaction (RT-PCR).
3. The percent release of LDH from all groups was measured spectrophotometrically.
4. The percent release of Cytochrome C was determined by immunocytochemical technique.
The results of the current study revealed that Doxorubicin and/or Doxycycline decreased the percentage of viable MCF-7 breast cancer cells in a concentration dependent manner, indicating that Doxorubicin and Doxycycline alone or in combination exhibit anti-proliferative effect against human breast cancer MCF-7 cell line and this effect was mediated through interference with normal cell cycle distribution and induction of apoptosis. Moreover, the results revealed that the combination of low dose Doxorubicin and Doxycycline generated synergistic anticancer effect in MCF-7 cells (CI=0.35). This suggests that Doxycycline may be an effective chemotherapeutic agent against breast cancer.
Additionally, we evaluated the effect of low dose Doxorubicin and/or Doxycycline on Bcl-2 gene expression in MCF-7 cells. The results revealed that Bcl-2 gene expression in cells treated with low dose Doxorubicin was insignificantly slightly higher than the control group but this effect was enhanced after combination with Doxycycline. Interestingly, it was found that Bcl-2 gene expression in MCF-7 cells treated with Doxycycline alone was significantly lower than the control group. These results suggested that Doxycycline either reduced Bcl-2 gene expression or may potentially interferes with Bcl-2 function and ultimately enhancing sensitivity of breast cancer cell line to Doxorubicin.
Moreover, a significant increase in the percent of LDH release from Doxycycline treated cells was observed in comparison to the untreated cells indicating the lethal effect of Doxycycline on the integrity of the cell membrane of MCF-7 cells. Meanwhile, the combination of both drugs also showed an elevation in the percent release of LDH with
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respect to the percent released from groups treated with either Doxorubicin or Doxycycline alone confirming the synergistic anticancer effect of the combination of both drugs on the viability of MCF-7 cells.
Furthermore, the results of this study demonstrated that the percent release of Cytochrome C from the mitochondria to the cytosol was insignificantly increased after treatment with a low dose Doxorubicin with respect to the control group, while on the other hand Doxycycline treatment showed significant release. However, addition of Doxycycline to a low dose Doxorubicin significantly increased the percent of Cytochrome C release suggesting that the apoptotic mitochondrial pathway of cell death, concerning Cytochrome C release, is involved.
These results suggested that Doxycycline shows cytotoxic effects on breast cancer MCF-7 cell line and ultimately enhanced sensitivity of these cells to low dose Doxorubicin. Thus, Doxycycline is an excellent example of how existing, inexpensive, well-tolerated drugs that can be repurposed as a new anticancer therapeutic agent.
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6.2. CONCLUSIONS
Doxorubicin and/or Doxycycline decreased the percentage of viable MCF-7 breast cancer cells in a concentration dependent manner.
The combination of low dose Doxorubicin and Doxycycline generated synergistic anticancer effect on MCF-7 cells (CI=0.35).
The growth inhibitory effect of small dose Doxorubicin may be partly via effects on Bcl-2 gene expression but it was obvious that this effect was enhanced after combination with Doxycycline.
Bcl-2 gene expression in MCF-7 cells was significantly decreased after treatment with a combination of a low dose Doxorubicin and Doxycycline. Moreover, Bcl-2 gene expression in cells treated with Doxycycline alone was significantly lower than that treated with a low dose of Doxorubicin alone.
The combination of low dose Doxorubicin and Doxycycline increased permeability of cell membrane, manifested by increase in the release of LDH from MCF-7 cells.
The release of Cytochrome C was also significantly increased after treatment with a combination of low dose Doxorubicin and Doxycycline.
Doxycycline may be an effective chemotherapeutic agent against breast cancer where the combination of low dose Doxorubicin and Doxycycline generated synergistic anticancer effect.
A low dose of Doxorubicin can effectively be combined with Doxycycline to enhance breast cancer treatment and minimize side effects. However, further studies to evaluate cell death mechanism as well as in vivo studies using animal models are required.
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6.3. RECOMMENDATIONS
In vivo experiments should be performed for confirmation of the obtained results.
Further work is required to explore whether Doxycycline may be a novel therapeutic drug useful for targeting breast cancer cells.