Electron transfer-based combination therapy of cisplatin with tetramethyl-p-phenylenediamine for ovarian, cervical, and lung cancers

Abstract

The platinum-based chemotherapy is the standard treatment for several types of cancer. However, cancer cells often become refractory with time and most patients with serious cancers die of drug resistance. Recently, we have discovered a unique dissociative electron-transfer mechanism of action of cisplatin, the first and most widely used platinum-based anticancer drug. Here, we show that the combination of cisplatin with an exemplary biological electron donor, N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), may overcome the resistance of cancer cells to cisplatin. Our steady-state absorption and fluorescence spectroscopic measurements confirm the effective dissociative electron-transfer reaction between TMPD and cisplatin. More significantly, we found that the combination of 100 μM TMPD with cisplatin enhances double-strand breaks of plasmid DNA by a factor of approximately 3.5 and dramatically reduces the viability of cisplatin-sensitive human cervical (HeLa) cancer cells and highly cisplatin-resistant human ovarian (NIH:OVCAR-3) and lung (A549) cancer cells. Furthermore, this combination enhances apoptosis and DNA fragmentation by factors of 2-5 compared with cisplatin alone. These results demonstrate that this combination treatment not only results in a strong synergetic effect, but also makes resistant cancer cells sensitive to cisplatin. Because cisplatin is the cornerstone agent for the treatment of a variety of human cancers (including testicular, ovarian, cervical, bladder, head/neck, and lung cancers), our results show both the potential to improve platinum-based chemotherapy of various human cancers and the promise of femtomedicine as an emerging frontier in advancing cancer therapy.

Fig. 1.

Spectroscopic observations of the DET reaction between cisplatin (CDDP) and TMPD. Absorption spectra (A–D) and fluorescence spectra (E, F) of TMPD in water or EtOH with and without the presence of CDDP. In B, spectra are also shown for the samples irradiated at a 266-nm laser with a power of 40 μW for 1 h, as well as the absorption spectrum of CDDP only.

Fig. 2.

Gel electrophoresis measurements of strand breaks in plasmid DNA treated by cisplatin alone and in combination with TMPD: (A) gel image, where the bands for supercoiled (SC) DNA, open circular DNA with single-strand breaks (SSBs), and linear DNA with double-strand breaks (DSBs) are indicated; (B) the DSB yield versus cisplatin concentration, obtained from the gel densitograms shown in Fig. S3 by integrating the DSB peak areas.

Fig. 3.

MTT cell viability assays of cancer cells with treatments of cisplatin alone and in combination with TMPD:(A) HeLa cells; (B) NIH:OVCAR-3 cells. The cells were treated by vehicle alone (0.2% EtOH),various concentrations of CDDP alone,and their combinations with 100 μM TMPD for 24 h. The values are represented as percent of viable cells, with the vehicle-treated cells regarded as 100% viable:mean of triplicate experiments (points); SD (bars). The expected additive cell viability curve (dashed line and Δ) was calculated by multiplying the effect of TMPD by that of CDDP at each concentration. Because the observed effect (green circle) is below that of the expected additive line, synergy was concluded.

Fig. 4.

Fluorescence microscopy observation of NIH:OVCAR-3 cells undergoing apoptosis induced by treatments indicated for 10 h. Hoechest 33342 staining (blue fluorescence) detects the nuclear fragmentation/condensation, while green fluorescence of FLICA reagent represents caspase activation. The images were acquired with a Nikon Eclipse TS100 microscope.

Fig. 5.

APO-BrdU DNA fragmentation assay of NIH:OVCAR-3 cells with treatments indicated for 24 h. Cells in the region above the line in each histogram are BrdU-positive cells (cells exhibiting DNA fragmentation); the line was drawn based on the samples not stained with any BrdU. Each percentage of BrdU-positive cells is the mean of triplicate experiments (standard deviation indicated).