Trichostatin A Enhances the Apoptotic Potential of Palladium Nanoparticles in Human Cervical Cancer Cells

Abstract

Cervical cancer ranks seventh overall among all types of cancer in women. Although several treatments, including radiation, surgery and chemotherapy, are available to eradicate or reduce the size of cancer, many cancers eventually relapse. Thus, it is essential to identify possible alternative therapeutic approaches for cancer. We sought to identify alternative and effective therapeutic approaches, by first synthesizing palladium nanoparticles (PdNPs), using a novel biomolecule called saponin. The synthesized PdNPs were characterized by several analytical techniques. They were significantly spherical in shape, with an average size of 5 nm. Recently, PdNPs gained much interest in various therapies of cancer cells. Similarly, histone deacetylase inhibitors are known to play a vital role in anti-proliferative activity, gene expression, cell cycle arrest, differentiation and apoptosis in various cancer cells. Therefore, we selected trichostatin A (TSA) and PdNPs and studied their combined effect on apoptosis in cervical cancer cells. Cells treated with either TSA or PdNPs showed a dose-dependent effect on cell viability. The combinatorial effect, tested with 50 nM TSA and 50 nMPdNPs, had a more dramatic inhibitory effect on cell viability, than either TSA or PdNPs alone. The combination of TSA and PdNPs had a more pronounced effect on cytotoxicity, oxidative stress, mitochondrial membrane potential (MMP), caspase-3/9 activity and expression of pro- and anti-apoptotic genes. Our data show a strong synergistic interaction between TSA and PdNPs in cervical cancer cells. The combinatorial treatment increased the therapeutic potential and demonstrated relevant targeted therapy for cervical cancer. Furthermore, we provide the first evidence for the combinatory effect and cytotoxicity mechanism of TSA and PdNPs in cervical cancer cells.

Keywords: apoptosis; caspases; cell viability; cervical cancer; mitochondrial membrane potential; oxidative stress; palladium nanoparticles; trichostatin A.

Figure 1

Synthesis and characterization of palladium nanoparticles (PdNPs). (A) Ultraviolet-visible spectroscopy (UV-VIS) spectra of PdNPs. Pdcl2: Palladium(II) chloride; (B) X-ray diffraction (XRD) pattern of PdNPs; (C) Fourier transform infrared spectroscopy (FTIR) spectra of PdNPs; (D) size distribution analysis of PdNPs by dynamic light scattering (DLS); (E) Transmission electron microscopy (TEM) images of PdNPs; (F) size distributions based on TEM images of PdNPs, ranging from 5–20 nm.

Figure 2

The dose-dependent effect of trichostatin A (TSA) and PdNPs on cell viability in human breast and cervical cancer cells. (A) The human breast cancer cells (MDA-MB-231) were incubated with various concentrations of TSA (0–300 nM) or PdNPs (0–300 nM), for 24 h. Cell viability was measured by WST-8; (B) Human cervical cancer cells were incubated with various concentrations of TSA (0–300 nM) or PdNPs (0–300 nM) for 24 h. Cell viability was measured using WST-8. The results are expressed as the mean ± standard deviation of three separate experiments. The treated groups showed statistically-significant differences from the control group, as determined by Student’s t-test (* p < 0.05).

Figure 3

Increasing concentrations of TSA or PdNPs enhance the loss of cell viability in human cervical cancer cells. (A) Human cervical cancer cells were co-incubated, for 24 h, with increasing concentrations of TSA (50–200 nM) and 50 nMPdNPs or increasing concentrations of PdNPs (50–200 nM) and 50 nM TSA (B). The results are expressed as the mean ± standard deviation of three separate experiments. The treated groups showed statistically-significant differences from the control group, as determined by the Student’s t-test (* p < 0.05). Con: Control.

Figure 4

The effect of TSA or PdNPs alone or the combinatorial effect of TSA and PdNPs on cell viability and HDAC activity, in human cervical cancer cells. Human cervical cancer cells were incubated with TSA (50 nM) or PdNPs (50 nM) or both TSA (50 nM) and PdNPs (50 nM) for 24 h. (A) Cell viability was measured using WST-8; (B) HDAC activity was measured.

Figure 5

The effect, of TSA, PdNPs or a combination of TSA and PdNPs, on human cervical cancer cell cytotoxicity. The cells were treated, for 24 h, with TSA (50 nM), PdNPs (50 nM) or a combination of TSA (50 nM) and PdNPs (50 nM). (A) Lactate dehydrogenase (LDH) activity was measured at 490 nm, using an LDH cytotoxicity kit (Aldrich, St. Louis, MO, USA); (B) Reactive oxygen species were measured, as the relative fluorescence of 2′,7′-dichlorofluorescein, with a spectrofluorometer. The results are expressed as the mean ± standard deviation of three independent experiments. The treated groups showed statistically-significant differences from the control group, as determined by Student’s t-test (* p < 0.05). NAC: N-acetylcysteine.

Figure 6

The effect of TSA, PdNPs or both TSA and PdNPs on oxidative stress markers, in human cervical cancer cells. Cells were treated for 24 h with TSA (50 nM), PdNPs (50 nM) or the combination of TSA (50 nM) and PdNPs (50 nM). (A) The concentration of malondialdehyde, expressed as nanomoles per milligram of protein; (B) the concentration of glutathione, expressed as milligram per gram of protein; (C) the specific activity of superoxide dismutase, expressed as units per milligram of protein; (D) the specific activity of catalase, expressed as units per milligram of protein. The results are expressed as the mean ± standard deviation of three independent experiments. There was a significant difference in the treated cells compared to the untreated cells, as determined by Student’s t-test (* p < 0.05).

Figure 7

The effect of TSA or PdNPs alone or a combination of TSA and PdNPs on mitochondrial membrane potential (MMP) and caspase-3 activities. Cells were treated for 24 h with TSA (50 nM), PdNPs (50 nM) or a combination of TSA (50 nM) and PdNPs (50 nM). (A) MMP (ratio of JC-1 aggregate to monomer) in cervical cancer cells was determined after treatment; (B) the cells were treated for 24 h with TSA (50 nM), PdNPs (50 nM) or a combination of TSA (50 nM) and PdNPs (50 nM), with and without caspase inhibitor. The concentration of p-nitroanilide released from the substrate was calculated from the absorbance at 405 nm. The results are expressed as the mean ± standard deviation of three separate experiments. The treated groups showed statistically-significant differences from the control group, as determined by Student’s t-test (* p < 0.05). ET: Etoposide.

Figure 8

Effect of TSA or PdNPs alone or in combination on apoptosis in human cervical cancer cells. The human cervical cancer cells were treated for 24 h with TSA (50 nM), PdNPs (50 nM) or a combination of TSA (50 nM) and PdNPs (50 nM). Apoptosis was assessed in a TUNEL assay; the nuclei were counterstained with DAPI. Representative images show apoptotic (fragmented) DNA (red staining) and the corresponding cell nuclei (blue staining).

Figure 8

Effect of TSA or PdNPs alone or in combination on apoptosis in human cervical cancer cells. The human cervical cancer cells were treated for 24 h with TSA (50 nM), PdNPs (50 nM) or a combination of TSA (50 nM) and PdNPs (50 nM). Apoptosis was assessed in a TUNEL assay; the nuclei were counterstained with DAPI. Representative images show apoptotic (fragmented) DNA (red staining) and the corresponding cell nuclei (blue staining).

Figure 9

The impact of TSA, PdNPs or a combination of TSA and PdNPs on the expression of apoptotic and anti-apoptotic genes. The relative mRNA expression of apoptotic and anti-apoptotic genes was analyzed by qRT-PCR, in human cervical cancer cells treated for 24 h with TSA (50 nM), PdNPs (50 nM) or a combination of TSA (50 nM) and PdNPs (50 nM). The results are expressed as the mean ± standard deviation of three separate experiments. The treatment groups showed statistically-significant differences from the control group, as determined by Student’s t-test (* p < 0.05).

Figure 9

The impact of TSA, PdNPs or a combination of TSA and PdNPs on the expression of apoptotic and anti-apoptotic genes. The relative mRNA expression of apoptotic and anti-apoptotic genes was analyzed by qRT-PCR, in human cervical cancer cells treated for 24 h with TSA (50 nM), PdNPs (50 nM) or a combination of TSA (50 nM) and PdNPs (50 nM). The results are expressed as the mean ± standard deviation of three separate experiments. The treatment groups showed statistically-significant differences from the control group, as determined by Student’s t-test (* p < 0.05).