Biochemical In-vitro Study of Tamoxifen Effect on CYP2D6 Enzyme and Cytotoxicity in Iraqi Premenopausal Women with Breast Cancer

Background:

 Hormone therapy is the first targeted therapy also called estrogen suppression therapy often used as an adjuvant therapy to reduce the risk of cancer back after surgery or cancer that has spread.  Tamoxifen is used to treat early breast cancer in women who have already been treated with surgery, radiation, and/or chemotherapy. It is used to reduce the risk of developing a more serious type of breast cancer in women.

Present study was planned to assess the effect of tamoxifen on cytochrome P4502d6 enzyme activity. As well, study the cytotoxic effect of tamoxifen on MCF-7 breast cancer cell line and survey the apoptosis mechanism for MCF-7.

Subjects and methods:

This study includes (60) pre-menopausal women, their age ranged between (25-48) years. Thirty subjects were apparently healthy, chosen as control group (C group) and sixty patients with breast cancer (P group), were divided into two groups according to type of treatment: thirty five patients under tamoxifen treatment (P1 group), and twenty five patients under fluoxetine and tamoxifen treatment (P2 group). The levels of all parameters (age, weight, height, BMI, waist, hip, WHR) were quantitatively determined in patients and control. The second part of this study include the determination of the cytotoxic effects of tamoxifen against MCF-7 breast cancer cell line and investigation of the mechanism by which the affected living cells toward apoptosis using Caspase -9 activity and High Content Screening (HCS) assay.

Results:

Part I:

• A significant increase (p˂0.01) in the mean of BMI was observed in the (P1) group and (P2) group in comparison with that of the group (C) (30.3 and 32.1 vs. 25.18 Kg/m²).
• The mean level of serum cytochrome P450 2d6 has significant increase in group (P1) and group (P2) in comparison with that in group (C) (32.2 and 19.43 vs. 13.87 U/L) while the result showed a significant decrease in the group (P2) when compared with group (P1)

Part II:

• Cytotoxic effects of tamoxifen against MCF-7 cell line showed half maximal inhibitory concentration IC₅₀ value was at 4.506µg/ml which is the most significant cytotoxic toward MCF-7 cell line treated for 24 hours.
• The effect of the tamoxifen on the mechanism of apoptosis reveal a highly increased caspase-9 activity, which observed at 5 µg/ml concentration in MCF-7 cell, IC₅₀ was at (4.506 µg/ml)
•  The effect of the tamoxifen on the mechanism of apoptosis showed a significant increase with increased concentration (dose dependent) for cell membrane permeability, cytochrome c and nuclear intensity at (100µg/ml of tamoxifen) when compared with (20µg/ml) of doxorubicin as a standard.
•   The effect of the tamoxifen on the mechanism of apoptosis showed a significant decrease when the concentration is increased (dose depended) for cell viability and mitochondrial membrane permeability at (100µg/ml) when compared with 20 µg/ml of doxorubicin as a positive control.
  

  Conclusions:

• In patients treated with tamoxifen, the measurement of CYP2D6 activity showed a good indicator of breast cancer
• Tamoxifen possess cytotoxic effect against breast cancer cells (MCF-7), as determined in-vitro by MTT assay (4.506 µg/ml).
• A toxic effect of tamoxifen has been demonstrated on MCF-7 cell line by using caspase-9 at IC₅₀ concentration (4.506 µg/ml)
• From HCS technique results; tamoxifen showed toxic effect toward MCF7 cell line at (100μg/ml) tamoxifen concentration in a dose-dependent manner with increase in cell membrane permeability, cytochrome C, nuclear intensity, and change in mitochondrial membrane potential and decrease in cell viability level.