Upregulation of apoptotic protease activating factor-1 expression correlates with anti-tumor effect of taxane drug.

Drug resistance is a multifactorial process involving a variety of mechanisms and genes. Taxane drug class like Docetaxel is not effective for all types' breast cancers and presents a huge clinical challenge. To improve cancer treatment outcome, it is important to distinguish which proteins can kill the cancer cells and whether the expression levels of these proteins affect treatment. Cancer cells are wildly known to be protected from apoptosis, due to low level of apoptotic protease activating factor-1 (Apaf-1) compared with normal cells. Apaf-1 is an essential protein that defines whether cytochrome c released form mitochondria remains stable or degrades. According to this hypothesis, increasing of Apaf-1 expression in MCF7 breast cancer cells was performed and Docetaxel efficacy examined. The immunoassay techniques were used to investigate Apaf-1 and cytochrome c levels, and different apoptosis assay methods applied to better understand the effect of Apaf-1 expression levels in cellular response to apoptotic stimuli by Docetaxel. Our results determined that cytoplasmic cytochrome c level elevated along with increasing Apaf-1 and MCF7 cells were sensitised to Docetaxel, suggesting that loss of Apaf-1 may cause Docetaxel-resistance in breast cancer cells through less apoptosome formation. ROS level increased in cells transfected with Apaf-1 and induced mitochondrial permeability for cytochrome c release, which subsequently promoted apoptosome formation, intrinsic apoptosis and ATP depletion.

Treating MCF7 breast cancer cell with proteasome inhibitor Bortezomib restores apoptotic factors and sensitizes cell to Docetaxel.

Chemoresistance is the leading cause of limiting long-term treatment success in cancer cells. Anticancer drugs usually kill cells through apoptosis induction and defects in this signaling pathway lead to chemoresistance. Apoptotic protease activating factor 1 regulates cellular stress evoked by chemotherapeutic agents through facilitating apoptosome assembling but can be degraded by proteasome. This study examined the role of proteasome inhibitor Bortezomib in the cytotoxic effects of Docetaxel on MCF7 cells response and its correlation with Apaf-1 expression level. MTT assay, caspase 3/7 activity assay, propidium iodide staining, adenosine triphosphate and reactive oxygen species amount measurements were utilized to demonstrate the role of Bortezomib in Docetaxel efficacy with and without Apaf-1 overexpressing. Meanwhile, two-dimensional cell migration assay was performed by scratch wound assay. The combination of Docetaxel with Bortezomib was significantly more cytotoxic compared single drug, more effectively delayed cell growth, reduced ATP level and increased ROS production. In Apaf-1 overexpressing, Docetaxel was more efficient in preventing cell migration, however, Docetaxel plus Bortezomib were not significantly effective; and fluorescence images supported the interpretation. Our findings demonstrated MCF7 resistance to Docetaxel is due in part to low Apaf-1 level and Apaf-1 overexpression resulted in the increase of cell susceptibility to Docetaxel stimulus. We assume that proteasome inhibitor may restore apoptotic proteins like Apaf-1 and prevent the degradation of cytosolic cytochrome c released by Docetaxel, consequently triggering intrinsic apoptosis and promoting cancer cell death. Collectively, treating MCF7 breast cells with proteasome inhibitor sensitizes cells to Docetaxel-induced apoptosis and possibly overcomes chemoresistance.

Effects of doxorubicin and docetaxel on susceptibility to apoptosis in high expression level of survivin in HEK and HEK-S cell lines as in vitro models.

A major problem in the cancer treatment is the inherent resistance to chemotherapy. Identifying proteins that, once introduced in cancer cells, lead to a decreased efficiency of treatment outcome constitutes a major goal for biomedical research and applications. Survivin is a protein of IAPs family which its high expression can be a potential candidate for regulating cell death and survival in cancer therapy. To investigate the association of survivin increment and resistance to drug, survivin-reconstituted HEK (HEK-S) and HEK cells were used as in vitro models for the doxorubicin and docetaxel cellular response. Both morphological observation and survival assay exhibited that survivin reconstitution cells were significantly resistant to apoptotic stimuli by both drugs. It was observed that survivin overexpression has led to a decrease in caspase 3/7 activity and ROS level of cells but an increase in ATP content. Also, survivin-reconstituted cell displayed less red fluorescence compared to control after stimulation by drugs. Moreover, wound healing assay showed the ability of survivin to cause neighbouring cells to increase resistance to induction. These findings demonstrated survivin could be a potential target that can be inhibited the function of different drugs with various mechanisms in chemotherapy.

Chitosan Immobilization on Bio-MOF Nanostructures: A Biocompatible pH-Responsive Nanocarrier for Doxorubicin Release on MCF-7 Cell Lines of Human Breast Cancer.

In this work, a bio-metal-organic framework (Bio-MOF) coated with a monodispersed layer of chitosan (CS; CS/Bio-MOF) was synthesized and applied as a pH-responsive and target-selective system for delivery of doxorubicin (DOX) in the treatment of breast cancer. The efficiency of the nanocarrier in loading and releasing DOX was assessed at different pH levels. To monitor the in vitro drug release behavior of the drug-loaded carrier, the carrier was immersed in a phosphate buffered saline solution (PBS, pH 7.4) at 37 °C. According to the observations, the nanocarrier presents a slow and continuous release profile as well as a noticeable drug loading capacity. In addition, the carrier demonstrates a pH-responsive and target-selective behavior by releasing a high amount of DOX at pH 6.8, which is indicative of tumor cells and inflamed tissues and releasing a substantially lower amount of DOX at higher pH values. In addition, the results indicated that pH is effective on DOX uptake by CS/Bio-MOF. A 3.6 mg amount of DOX was loaded into 10 mg of CS/Bio-MOF, resulting in a 21.7% removal at pH 7.4 and 93.0% at pH 6.8. The collapsing and swelling of the CS layers coated on the surface of the Bio-MOFs were found to be responsible for the observed pH dependence of DOX delivery. Moreover, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the trypan blue test were performed on the MCF-7 (breast cancer) cell line in the presence of the CS/Bio-MOF carrier to confirm its biological compatibility. In addition, Annexin V staining was conducted to evaluate the cytotoxicity of the free and loaded DOX molecules. On the basis of the obtained optical microscopy, MTT assay, fluorescence microscopy, and dyeing results, the CS/Bio-MOF carrier greatly enhances cellular uptake of the drug by the MCF-7 cells and, therefore, apoptosis of the cells due to its biocompatibility and pH-responsive behavior.