Carboplatin and oxaliplatin in sequenced combination with bortezomib in ovarian tumour models

Abstract

Background: Ovarian cancer remains an on-going challenge mainly due to the development of drug resistance and also because the cancer is likely to have metastasized at the time of diagnosis. Currently, chemotherapy based on platinum drugs such as cisplatin is the primary treatment for the disease. Copper transporter 1 is involved in the transport of cisplatin into the cell, but is also down-regulated by the drug. Bortezomib, a proteasome inhibitor, has been reported to block this platinum-induced down-regulation of CTR1, so that in the presence of bortezomib, the cellular uptake of platinum drugs may be increased. Increased platinum accumulation may result in increased platinum - DNA binding so that the platinum drug in combination with bortezomib may produce enhanced cell kill.

Methods: In this study the efficacy of the sequential combinations of carboplatin, oxaliplatin and a trans-platinum compound coded as CH1 with BORT on the human ovarian A2780, A2780cisR, A2780ZD0473R and SKOV-3 cancer cell lines was evaluated. The levels of cellular platinum accumulation and platinum-DNA binding were determined following the treatment with these combinations. In order to investigate the effect of the combinations of the formation of ROS, the total and oxidized glutathione levels were also determined.

Results: Prevention of copper transporter 1 degradation by bortezomib is found to enhance the cellular accumulation of platinum, the level of Platinum - DNA binding and increases oxidative stress especially in the resistant cell lines.

Conclusions: The results suggest that the prevention of CTR1 degradation by bortezomib may be playing a major role in increasing the cellular uptake of platinum drugs and platinum-DNA binding level. Furthermore, the generation of oxidative stress appears to be a major contributor to the enhanced cell kill.

Figure 1

Simplified representation of cellular events responsible for apoptosis due to combination of platinum drugs and bortezomib in which DNA damage, ER stress and increase in ROS due to platinum drugs and inactivation of antiapoptotic proteins such as NF-kB by bortezomib lead to apoptosis. L1 and L2 denote the carrier ligands and L3 and L4 denote the leaving groups in CS, CB and OX. In CH1 which has a trans-geometry, L1 and L4 represent the carrier ligands and L2 and L3 represent the leaving groups.

Figure 2

Chemical structures of platinum compounds carboplatin (CB), oxaliplatin (OX), trans-bis(3-hydroxypyridine)dichloroplatinum(II) (CH1), and that of proteasome inhibitor bortezomib (BORT), applied in combination to ovarian cancer cell lines.

Figure 3

Inhibition of cell growth as a function of increasing concentrations of a) CB, b) OX c) CH1 and d) BORT as applied to the ovarian cancer cell lines A2780, A2780^cisR, A2780^ZD0473R and SKOV-3. Cell survival fractions following treatment with increasing concentrations of drugs for 72 h were determined using MTT assay and spectrophotometric analysis. Error bars represent the standard deviation.

Figure 4

Combination Index (CI) values applying to the sequenced combinations of a) CB, b) OX and c) CH1 with BORT administered to the ovarian A2780, A2780^cisR, A2780^ZD0473R and SKOV-3 cancer cell lines. CI values were calculated following 72 h treatments with the drugs at their equipotent ratios. CI values of <1, =1 and >1 indicate respectively synergism, additivity and antagonism in combined drug action.

Figure 5

Cellular platinum accumulation in ovarian cancer A2780 and A2780^cisR, A2780^ZD0473R and SKOV-3 cell lines as applied to single treatments of CB and OX and their selected combinations with BORT. Cells were treated with the drugs for 24 h followed by collection, lysis and finally the detection of Pt was using AAS. Data were statistically analyzed using the paired Student’s t test: * p <0.05 indicates significant difference from control. Error bars represent the standard deviation.

Figure 6

Platinum − DNA binding levels in the ovarian A2780, A2780^cisR, A2780^ZD0473R and SKOV-3 cancer cell lines due to treatment with CB and OX and their combinations with BORT. Cells were treated with the drugs for 24 h followed by collection, DNA extraction and finally the detection of Pt using AAS. Data were statistically analyzed using the paired Student’s t test: * p <0.05 indicates significant difference from control. Error bars represent the standard deviation.

Figure 7

Levels of reduced (GSH) and oxidized (GSSG) forms of cellular glutathione in relative luminescence units (RLU × 10⁵) before and after treatments with BORT alone and its combination with CB and OX in the ovarian cancer A2780, A2780^cisR and SKOV-3 cell lines. Cells were treated for 24 h and glutathione content was determined using GSH/GSSG-Glo Assay kit. Data were statistically analyzed using the paired Student’s t test: * p <0.05 indicates significant difference from control. Error bars represent the standard deviation.