Pentoxifylline sensitizes human cervical tumor cells to cisplatin-induced apoptosis by suppressing NF-kappa B and decreased cell senescence

Abstract

Background: Worldwide, cervical cancer is the second most common causes of cancer in women and represents an important mortality rate. Cisplatin (CIS) is a very important antitumoral agent and can lead tumor cells toward two important cellular states: apoptosis and senescence. In some types of cancers pentoxifylline (PTX) sensitizes these cells to the toxic action of chemotherapeutics drugs such as adriamycin, inducing apoptosis. In the present work, we studied in vitro whether PTX alone or in combination with CIS induces apoptosis and/or senescence in cervix cancer HeLa and SiHa cell lines infected with HPV types 16 and 18, respectively, as well as in immortalized keratinocytyes HaCaT cells.

Methods: HeLa (HPV 18+), SiHa (HPV 16+) cervix cancer cells and non-tumorigenic immortalized HaCaT cells (control) were treated with PTX, CIS or both. The cellular toxicity and survival fraction of PTX and CIS were determinate by WST-1 and clonogenic assays respectively. Apoptosis, caspase activation and phosphorylation of ERK1/2, p38, p65 (NF-κB), Bcl-2 and Bcl-XL anti-apoptotic proteins were determinated by flow cytometry. Senescence by microscopy. Phosphorylation of IκBα and IκB total were measured by ELISA. Pro-apoptotic, anti-apoptotic and senescence genes, as well as HPV-E6/7 mRNA expression, were detected by RT-PCR.

Results: Our results show that after 24 hours of incubation PTX per se is toxic for cancer cells affecting cell viability and inducing apoptosis. The toxicity in HaCaT cells was minimal. CIS induces apoptosis in HeLa and SiHa cells and its effect was significantly increases when the cells were treated with PTX + CIS. In all studies there was a direct correlation with levels of caspases (-3, -6, -7, -9 and -8) activity and apoptosis. CIS induces important levels of senescence and phosphorylation of ERK1/2, p38, p65/RELA, and IκBα, and decreased the expression of anti-apoptotic protein Bcl-XL. Surprisingly these levels were significantly reduced by PTX in tumor cells, and at the same time, increases the expression of pro-apoptotic genes.

Conclusion: PTX sensitizes cervical cancer cells to CIS-induced apoptosis and decreases the CIS-induced senescence in these cells via inhibition of NF-κB signaling pathway; diminishes expression of antiapoptotic proteins and the activation of caspases.

Figures

Figure 1

Determination of late apoptosis and caspase activity of HeLa, SiHa and HaCaT cells after in vitro treatment with pentoxifylline or cisplatin either alone or in combinations. 24 hours later the cells were harvested and late apoptosis was determined by UV light microscopy using ethidium bromide and acridine orange stains, the results represent late apoptosis index (Figure 1A). Caspases-3, -6, - 7, -9 and -8 activation was determined by flow cytometry, the results represent the percentage of caspase activity (Figure 1B and 1C respectively). The results represent the mean ± SD of three independent experiments carried out in triplicate. Statistical analysis, Student's t test. (*) P < 0.001 vs CTL. (♦) P < 0.001 vs CIS.

Figure 2

Determination of β-galactosidase-associated senescence of HeLa, SiHa and HaCaT cells after in vitro treatment with PTX or CIS either alone or in combinations. 24 hours later the cells were harvested and senescence was determined by histochemistry using senescence detection kit (BioVision Mountain View, CA, USA). The results represent the mean ± SD of three independent experiments carried out in triplicate. Statistical analysis, Student's t test. (*) P < 0.001 vs CTL. (♦) P < 0.001 vs CIS.

Figure 3

Phosphorylation of the IκBα [pS32] and IκBα (Total) by ELISA kit of HeLa and SiHa cells after in vitro treatment with pentoxifylline or cisplatin either alone or in combination. 24 hours later the cells were harvested and the phosphorylation of the IκBα [pS32] and IκBα (Total) was determined by commercial ELISA kit (Invitrogen). The results represent the mean ± SD of three independent experiments carried out in triplicate. Statistical analysis Student's t test. (*) P <0.001 vs CTL. (•) P <0.001 vs TNF-α. (♦) P <0.001 vs CIS.

Figure 4

Determination of phosphorylated ERK 1/2, p38, and p65 in HeLa, SiHa and HaCaT cell treatment with pentoxifylline or cisplatin either alone or in combination. 24 hours later the cells were harvested and the phosphorylated ERK1/2, p38 and p65 proteins were determined by flow cytometry. A total of 20,000 events were registered in each test. The results represent the mean ± SD of 3 independent experiments carried out in triplicate. (*) P <0.001 vs untreated control cells. (♦) = P <0.001 vs CIS.

Figure 5

Determination of Bcl-2 and Bcl-XL anti-apoptotic proteins in cervical tumor cells treated with PTX or CIS either alone or in combination. 24 hours later the cells were harvested and the proteins expression were determined by flow cytometry. A total of 20,000 events were registered in each test. The results represent the mean ± SD of 3 independent experiments carried out in triplicate. (*) = P < 0.01 vs CTL. (♦) P < 0.05 vs CIS.

Figure 6

Changes in the expression of caspases, senescence, NF-κB, pro- and antiapoptotic-related genes after in vitro exposure to pentoxifylline or cisplatin either alone or in combination. The gene expressions were determined by real-time quantitative PCR. The data are expressed as mRNA fold-increase using mRNA ribosomal as a reference gene. Experiments were conducted in triplicates and repeated three times. In all cases, SD was not > 0.08.