Pentoxifylline Sensitizes Cisplatin-Resistant Human Cervical Cancer Cells to Cisplatin Treatment: Involvement of Mitochondrial and NF-Kappa B Pathways

Abstract

Background: Cervical cancer continues to be a major public health problem worldwide, and Cisplatin is used as first-line chemotherapy for this cancer; however, malignant cells exposed to CISplatin (CIS) become insensitive to the effects of this drug. PenToXifylline (PTX) is a xanthine that sensitizes several types of tumor cells to apoptosis induced by antitumor drugs, such as Adriamycin, Carboplatin, and CIS. The effects of PTX on tumor cells have been related to the disruption of the NF-κB pathway, thus preventing the activation of cell survival mechanisms such as the expression of anti-apoptotic genes, the secretion of proinflammatory interleukins, and growth factors.

Objective: In this work, we studied the antitumor proprieties of PTX in human SiHa cervical carcinoma cells resistant to CIS.

Materials and methods: SiHa and HeLa cervical cancer cells and their CIS-resistant derived cell lines (SiHaCIS-R and HeLaCIS-R, respectively) were used as in-vitro models. We studied the effects of PTX alone or in combination with CIS on cell viability, apoptosis, caspase-3, caspase-8, and caspase-9 activity, cleaved PARP-1, anti-apoptotic protein (Bcl-2 and Bcl-xL) levels, p65 phosphorylation, cadmium chloride (CdCl₂) sensitivity, Platinum (Pt) accumulation, and glutathione (GSH) levels, as well as on the gene expression of GSH and drug transporters (influx and efflux).

Results: PTX sensitized SiHaCIS-R cells to the effects of CIS by inducing apoptosis, caspase activation, and PARP-1 cleavage. PTX treatment also decreased p65 phosphorylation, increased Pt levels, depleted GSH, and downregulated the expression of the ATP7A, ATP7B, GSR, and MGST1 genes.

Conclusion: PTX reverses the acquired phenotype of CIS resistance close to the sensitivity of parental SiHa cells.

Keywords: NF-KappaB; cervical cancer cells; chemoresistance; cisplatin; pentoxifylline.

Figure 1

PTX decreases viability and induced sensitization to CIS treatment in resistant cervical cancer cell lines. Cell viability was assessed using the WST-1 assay (A). HeLaP and HeLaCIS-R cell lines were treated with CIS (20 μM), PTX (4 mM), or PTX + CIS (4 mM + 20 μM) for 24 h (B). SiHaP and SiHaCIS-R cells were treated with CIS (30 μM), PTX (4 mM), or PTX + CIS (4 mM + 30 μM) for 24 h. Data are expressed as the percentage of cells relative to the UCG. *p < 0.01 = statistical significance after comparison among the PTX, or CIS, or PTX + CIS groups in HeLaCIS-R cells or SiHaCIS-R vs. parental cells. ● p < 0.01 PTX + CIS vs. PTX group or CIS group in parental or resistant cells. UCG, Untreated Control Group; CIS, Cisplatin; PTX, PenToXifylline.

Figure 2

PTX-induced apoptosis and caspase activities in SiHaP and SiHaCIS-R cells. SiHaP and SiHaCIS-R cells were treated with CIS (30 μM), PTX (4 mM), or PTX + CIS (4 mM + 30 μM) for 24 h; then, apoptosis (DNA fragmentation (A), caspase-9 (B); caspase-8 (C), and caspase-3 (D) activity were determined. Data are expressed as fold change relative to the UCG. *p < 0.001 = statistical significance after comparison among PTX, or PTX + CIS groups vs. CIS or UCG in SiHaP cell or SiHaCIS-R cells. +p < 0.001 CIS group vs. UCG in SiHaP cells. ● p < 0.001 PTX + CIS group vs. PTX in SiHaCIS-R cells. UCG, Untreated Control Group; CIS, CISplatin; PTX, PenToXifylline.

Figure 3

PTX in combination with CIS increased PARP-1 cleavage in chemoresistant SiHa cells. Western blot analysis of PARP-1 in SiHaP and SiHaCIS-R cells were treated with CIS (30 μM), PTX (4 mM), or PTX + CIS for 24 h. Then, PARP cleavage was evaluated by Western blot (A). Densitometric analysis of total PARP-1 (B) and the cleavage of PARP (C) are shown. Relative density was calculated using Image Studio Lite ver. 5.2.5 (LI-COR Biotechnology) software. A representative example of three assays performed in triplicate is shown. β-Actin was used as a loading control. UCG, Untreated Control Group; CIS, CISplatin; PTX, PenToXifylline.

Figure 4

PTX decreased NF-κB/p65 phosphorylation and Bcl-2 and Bcl-XL anti-apoptotic proteins in SiHaP and SiHaCIS-R cells. SiHaP and SiHaCIS-R cells were treated with CIS (30 μM), PTX (4 mM), or PTX + CIS (4 mM + 30 μM) for 24 h to determine Bcl-XL and Bcl-2 expression. For the assessment of p65 phosphorylation, the cells were treated for 1 h. Data are expressed as the Mean Fluorescence Intensity (MFI) of p65 phosphorylation (A, B), Bcl-XL (C, D), and Bcl-2 (E, F) anti-apoptotic proteins. *p < 0.001 = statistical significance after the comparison between PTX group or the PTX + CIS group vs. CIS group or UCG groups in SiHaP cells or SiHaCIS-R cells. ● p < 0.001 CIS group vs. UCG group in SiHaP cells or SiHaCIS-R cells. UCG, Untreated Control group; CIS, CISplatin; PTX, PenToXifylline.

Figure 5

PTX decreased glutathione levels induced by CIS in parental and chemoresistant SiHa cells. SiHaP and SiHaCIS-R cells were treated with CIS (30 μM), PTX (4 mM), or CIS + PTX for 24 h; then, the glutathione levels were determined. Data are expressed as ng of GSH/10⁶ cells. *p < 0.001 = statistical significance after the comparison between PTX group or PTX + CIS group vs. CIS group or UCG in SiHaCIS-R cells. + p < 0.001 PTX group or PTX + CIS group vs. CIS group in SiHaP cells. **p < 0.001 PTX + CIS group vs. PTX group in SiHaCIS-R cells. ● p < 0.001 CIS group vs. UCG group in SiHaCIS-R or SiHaP cells. UCG, Untreated Control Group; CIS, CISplatin; PTX, PenToXifylline.

Figure 6

PTX downregulated efflux pump, multidrug resistance, and glutathione genes in SiHaP and SiHaCIS-R cells (A, B). SiHaP and SiHaCIS-R cells were treated with CIS (30 μM), PTX (4 mM), or PTX + CIS (4 mM + 30 μM) for 4 h. Efflux (ATP7A, ATP7B, and MRP-2), influx (CTR1), and Glutathione genes (GRS, GSS, GPX, and MGST1) were evaluated by qPCR. RPL32 was used as a reference gene. *p < 0.05 = statistical significance, comparisons were performed between PTX or PTX + CIS vs. CIS or UCG groups. Data are expressed as fold changes relative to the UCG of SiHaP cells (C, D). Heat Maps of efflux (ATP7A, ATP7B, and MRP-2), influx (CTR1), and glutathione genes (GRS, GSS, GPX, and MGST1) expressed in SiHaP and SiHaCIS-R cells treated with PTX, CIS, or PTX + CIS.