Sensitization of cervix cancer cells to Adriamycin by Pentoxifylline induces an increase in apoptosis and decrease senescence
- Alejandro Bravo-Cuellar1, 2,
- Pablo C Ortiz-Lazareno1,
- Jose M Lerma-Diaz1,
- Jorge R Dominguez-Rodriguez1,
- Luis F Jave-Suarez1,
- Adriana Aguilar-Lemarroy1,
- Susana del Toro-Arreola3,
- Ruth de Celis-Carrillo1,
- Jose E Sahagun-Flores3, 5,
- Javier E Garcia de Alba-Garcia4 and
- Georgina Hernandez-Flores1Email author
© Bravo-Cuellar et al; licensee BioMed Central Ltd. 2010
Received: 25 June 2009
Accepted: 19 May 2010
Published: 19 May 2010
Chemotherapeutic drugs like Adriamycin (ADR) induces apoptosis or senescence in cancer cells but these cells often develop resistance and generate responses of short duration or complete failure. The methylxantine drug Pentoxifylline (PTX) used routinely in the clinics setting for circulatory diseases has been recently described to have antitumor properties. We evaluated whether pretreatment with PTX modifies apoptosis and senescence induced by ADR in cervix cancer cells.
HeLa (HPV 18+), SiHa (HPV 16+) cervix cancer cells and non-tumorigenic immortalized HaCaT cells (control) were treated with PTX, ADR or PTX + ADR. The cellular toxicity of PTX and survival fraction were determinated by WST-1 and clonogenic assay respectively. Apoptosis, caspase activation and ADR efflux rate were measured by flow cytometry, senescence by microscopy. IκBα and DNA fragmentation were determinated by ELISA. Proapoptotic, antiapoptotic and senescence genes, as well as HPV-E6/E7 mRNA expression, were detected by time real RT-PCR. p53 protein levels were assayed by Western blot.
PTX is toxic (WST-1), affects survival (clonogenic assay) and induces apoptosis in cervix cancer cells. Additionally, the combination of this drug with ADR diminished the survival fraction and significantly increased apoptosis of HeLa and SiHa cervix cancer cells. Treatments were less effective in HaCaT cells. We found caspase participation in the induction of apoptosis by PTX, ADR or its combination. Surprisingly, in spite of the antitumor activity displayed by PTX, our results indicate that methylxantine, per se does not induce senescence; however it inhibits senescence induced by ADR and at the same time increases apoptosis. PTX elevates IκBα levels. Such sensitization is achieved through the up-regulation of proapoptotic factors such as caspase and bcl family gene expression. PTX and PTX + ADR also decrease E6 and E7 expression in SiHa cells, but not in HeLa cells. p53 was detected only in SiHa cells treated with ADR.
PTX is a good inducer of apoptosis but does not induce senescence. Furthermore, PTX reduced the ADR-induced senescence and increased apoptosis in cervix cancer cells.
Cervix cancer is the most frequently diagnosed female cancer in developing countries and the second most frequent cancer affecting women worldwide . An estimate of half a million new cases in 2008 were reported . The most important risk factor in this cancer is the presence of human papilloma virus (HPV) infection. High risk HPV types 16 and 18 are responsible for over 70% of cases of cervix cancer .
Cervix cancer, like other tumors shows two critical cellular stages: apoptosis and senescence. The first one occurs during normal or physiological conditions or by stimuli such as chemotherapy and constitutes a common pathway for cell replacement, tissue remodeling, damaged cell removal and elimination of cancer cells [4–6]. It is a complex process which involves caspases participation, activation of proapoptotic genes, among other molecules. Apoptosis is defined by morphologic features which include cell membrane blebbing, cell shrinkage, chromatin condensation, and nucleosomal fragmentation [7, 8]. Cellular senescence, originally defined as a phenotype of arrested cells, after a certain number of cell divisions. Now is considered a general biological program of terminal growth arrest, and can be induced by the shortening of telomeres (growing old) or by injuries to DNA which do not involve telomere shortening (accelerated senescence) [9, 10]. In this state, while they may remain metabolically active, cells can not divide even if they are stimulated by mitogens. They can be distinguished morphologically by their enlarged and flattened cell shape and increased granularity. This distinction is identifiable with considerable specificity by detection of β-galactosidase (SA-β-gal) by X-gal activity staining. Senescence shows a dual role in cancer patients. Since this process inhibits tumor cell proliferation it was considered to be a protection mechanism. However, recent data suggest that it also facilitates cancer progression [9–11].
Patients with advanced, persistent, or recurrent squamous cell carcinoma are usually treated with cytotoxic chemotherapeutic agents such as Adriamycin (ADR) which kills cancer cell mainly by apoptosis . This drug can also induce senescence [12, 13]; however, tumor cells can develop resistance to chemotherapy and generate responses of short duration or complete failure . Molecular basis of resistance to cancer therapy is not well understood. It is considered that several factors can play a role. Among these mechanisms, the transcriptional nuclear factor-κB (NF-κB) is a key regulator of genes involved in cellular proliferation, secretion of soluble factors such as TNFα and up-regulation of antiapoptotic genes [15–19].
Pentoxifylline (PTX), [1-(5-oxohexyl) 3, 7,-dimethylxanthine], is a nonspecific phosphodiesterase inhibitor which has been already clinically and routinely used for circulatory diseases for more than twenty years, and it is a potent NF-κB and TNFα inhibitor. Recently, PTX has been used to sensitize tumor cells to chemo- and radio-therapy. In our experience, we have observed that lymphoma-bearing mice treated with PTX + ADR survived more than one year after receiving only half of the standard therapeutically active ADR dose [20, 21]. PTX also sensitizes leukemic cells to perillyl alcohol-induced apoptosis  and also to prostatic tumor, HeLa and hepatoma cell line .
Cervix cancer is a public health problem and chemotherapy is not actually effective. Additionally, the effectiveness of chemotherapy has been limited by its side effects and/or the toxicity of these drugs in normal tissues and normal cells. Thus, the use of pharmacological agents that act by inhibiting resistance mechanisms or sensitizing cancer cells to chemotherapy without increasing side effects, are strongly desirable. We attempted to study in vitro whether pretreatment with PTX modifies apoptosis and senescence induced by ADR in cervix cancer cell lines.
Cell viability of human cervix cancer cells after in vitro treatment with PTX or ADR either alone or in combination
Viability of HeLa, SiHa or HaCaT cells after in vitro exposition to Pentoxifylline and Adriamycin
CELL LINE PERCENTAGE OF CELLULAR VIABILITY ± SD
PTX 8 mM
63.6 ± 2.1
57.8 ± 1.0
74.1 ± 1.0
ADR 1 μM
88.6 ± 3.0
70.1 ± 2.0
95.0 ± 7.6
PTX + ADR
40.2 ± 1.0
33.0 ± 1.2
78.5 ± 1.1
Clonogenic assay in HeLa, SiHa and HaCaT cells after in vitro treatment with PTX or ADR either alone or in combination
Pentoxifylline blocks Adriamycin efflux
Early apoptosis induced by PTX alone or in combination with ADR
Early apoptosis of HeLa, SiHa or HaCaT cells
(% mean ± SD)
(% mean ± SD)
(% mean ± SD)
3.7 ± 1.0
2.7 ± 0.2
10.0 ± 1.5
PTX 8 mM
30.5 ± 1.1 *
28.7 ± 1.0 *
30.3 ± 2.3 *
ADR 1 M
23.3 ± 2.1 *
28.6 ± 1.8 *
20.0 ± 1.7 *
PTX + ADR
38.4 ± 1.6 *
43.3 ± 2.5 *
31.0 ± 1.5 *
(% mean ± SD)
(% mean ± SD)
(% mean ± SD)
3.7 ± 1.0
2.5 ± 1.0
10.0 ± 1.5
PTX 8 mM
28.4 ± 2.1 *
19.0 ± 1.2 *
20.5 ± 1.8 *
ADR 1 M
27.0 ± 1.5 *
33.0 ± 3.0 *
17.5 ± 2.7 *
PTX + ADR
41.5 ± 1.0 *
45.4 ± 2.6 *
30.5 ± 1.5 *
(% mean ± SD)
(% mean ± SD)
(% mean ± SD)
3.8 ± 0.2
3.3 ± 0.2
10.0 ± 1.5
PTX 8 mM
6.0 ± 0.7
6.2 ± 1.2
10.3 ± 1.8
ADR 1 M
15.5 ± 1.0 *
6.3 ± 0.9
10.2 ± 1.7
PTX + ADR
23.1 ± 1.4 *
5.5 ± 0.8
15.4 ± 1.5 *
Late apoptosis induced by PTX, ADR or their combination
PTX and ADR induce caspase activation in HeLa and SiHa cells
PTX reduces ADR-induced senescence in cervix cancer cells
PTX increases IκBα protein
p53 protein expression and mRNA E6/E7 in HeLa and SiHa cells treated with PTX, ADR or PTX + ADR
Oncoproteins E6 and E7 play a very important role because they show close relation with p53 regulation. For this reason were studied, in the same experimental conditions, the mRNA expression in both cervical tumor cell lines. In Figure 7b we can see that in HeLa cells, PTX increased the expression of E6 mRNA (Δ% = 28). The treatment with ADR induced a contrary effect in an important fashion (Δ% = -84) and the experimental group treated with PTX + ADR increased E6 mRNA expression in HeLa cells (HPV 18) with a Δ% = 33. In relationship to E7 we observed practically a mirror behavior of E6 in all treatments. In the same way we evaluated E6 and E7 in SiHa cells (HPV 16). PTX did not modify mRNA expression of E6 (Δ% = 4) with ADR or PTX + ADR was strictly similar between themselves and lower than the control group (Δ% = -38 and -44 respectively). For E7 from HPV 16 we observed an important down regulation being most important in PTX + ADR group (PTX Δ% = -52, ADR Δ% = -43, PTX + ADR Δ% = -61).
mRNA expression of proapoptotic, antiapoptotic and senescence genes in cervix tumor cell lines after in vitro treatment with PTX alone or in combination with ADR
In SiHa cells treated with PTX, we found 0.3 to 3 fold up-regulation in p16, p21, caspase-3, moreover caspase-9 was 4 fold up-regulated (Figure 8). When SiHa cells were treated with ADR we observed 0.3 to 3 fold up-regulation of p21, diablo, caspase-3 and caspase-9, p16 genes. In SiHa cells treated with PTX + ADR, we observed 0.3 to 3 fold up-regulation of puma, diablo, caspase-3 and p16. However the up-regulation was greater in caspase-9, p21 and noxa more than 3.7-fold increase (Figure 8). We also found up-regulation in bad, bax, bak, noxa, puma, p21, and diablo gene in HaCaT cells treated with PTX + ADR. It was especially significant in puma and p21 genes being above a 10 fold increase. On the contrary antiapoptotic genes bcl- XL , mcl-1, and survivin were down-regulated or not modified in practically all cellular lines by the different treatments, with the exception of survivin gene in HeLa cells (1.6 fold increase) treated with PTX and mcl-1 in HeLa cells (0.8 fold increase) treated with PTX + ADR. These data suggested the up-regulation in favour of genes with proapoptotic activity principally in HeLa and SiHa cancer cells treated with PTX + ADR.
This work shows that PTX per se may induce apoptosis of cervix cancer cell lines but does not induce senescence. In the same manner, but no less important, PTX sensitizes these cells to the induction of apoptosis by ADR and at the same time reduces significantly the senescence caused by this anthracycline.
Infections with high-risk HPV are the predominant associated risk factors in the development of cervical cancer. In addition, proteins E6 and E7 expressed by HPV contribute to resistance to treatments . We used HeLa and SiHa cells infected with HPV 18 and 16 respectively to be closer to the most common HPV high risk types observed in patients.
The studies of toxicity in cervix tumor cells, including clonogenic assay show that PTX has antitumoral activity by itself and the most important results are observed when the cells are pretreated with PTX before ADR. All these results strongly suggest that the combination of PTX + ADR not only induced death in tumor cells, but also reduced the number of cells with reproductive capacity after treatment.
Early and late apoptosis were also induced more efficiently by the combination of the drugs in the two cervix cancer cell lines. Our results are in agreement with previous experiments and with the published data , suggesting that the sensitizing effect of the PTX is due to a general mechanism, present in different cell lines and not in a particular one, even if the cells are infected with high risk HPV.
To explain the differences in late apoptosis in HeLa and SiHa culture treated with ADR, it is important consider that late apoptosis it is not reversible and each system could differ depending the genetic background of the cells. For example HeLa cells have 50 integrated copies of the HPV 18 whereas SiHa cells have only two copies of the HPV 16 and its rate of replication is very different .
The effect of PTX alone or in combination with ADR has been related to caspase activation [21, 26]. At this point it is necessary to stress that in general our results are in agreement with other experiments reported here, so the higher or lower activity of caspases was correlated with higher or lower percentage of apoptosis.
Senescence originally was defined as cell arrest growth , and considered a novel tumor suppressive mechanism . However, the role of senescence in Oncology has to be enlightened. It is true that the senescent cells can not replicate itself, but it can stimulate the growth and dissemination of other tumor cells by secretion of different products like growth factors, cytokines and metalloproteases. It has been reported that senescent cells, induce an increment in the cell proliferation of precancerous cells [28, 29]. To our knowledge, this is the first report indicating that PTX alone can induce apoptosis but does not induce a senescence cell state. In addition, it is also very important to remark that PTX pretreatment significantly reduces ADR-induced senescence and in turn increases the apoptotic index in HeLa and SiHa cancer cells. Our observations support also the idea that independently of the type of HPV virus that infects these cells, they can undergo apoptosis which is preferable, to cellular senescence.
These results can be explained biochemical and biologically by different facts: PTX is an effective inhibitor of phosphodiesterase (PD) activity (mainly PD4), and has also been reported to prevent the arrival of the NF-κB to the cellular nucleus by inhibition of the phosphorylation of serine 32 and 36 of the IκB complex in lymphoma and the human monocyte cell line U937 [21, 24]. In this sense, we find that PTX alone or in combination with ADR causes an increment of IκBα in HeLa and SiHa cells. This accumulation of IκBα induced by PTX could lead to a significant retention and therefore less activation of NF-κB, which in turn suppresses survival signals induced by this factor.
Our results show that PTX decreases ADR efflux in HeLa and SiHa cells and this increases the ADR cytotoxic effect. It has been reported that PTX down-regulated mdr1 gene expression and avoid ADR efflux, and in turn, therefore decrease the resistance of L1210 leukemic cells to ADR effects .
This methylxanthine arrests the cell cycle in the G2 phase, in which tumor cells are more sensitive to the toxic effects of some chemotherapeutic agents and radiotherapy [22, 31]. ADR can induce reactive oxygen species which are described as apoptosis or senescence inductors . PTX has been described as an antioxidant molecule  and this may explain why PTX alone does not induce senescence but impairs the senescence induced by ADR.
Using Real-Time PCR assay, we studied the mRNA expression of genes related with induction of apoptosis, caspases and senescence. In general we observed an up-regulation of proapoptotic genes. The most important up-regulation is observed in caspase-3 and caspase-9, noxa, puma, and diablo and down-regulation in antiapoptotic family genes in HeLa and SiHa cells treated with PTX, ADR or both.
We observed up-regulation in p53 gene in HeLa cells and in some groups of SiHa cells. The p53 protein was not found except in SiHa cells treated exclusively with ADR. This could indicate that apoptosis by PTX is p53-independent and suggests the participation of other molecules such as caspase-3 and caspase-9 as observed in this study. The up-regulation of the puma gene and p53 protein abrogation in these cells treated with PTX lets us to suppose that multiples pathways may have been involved to induce puma, such as endoplasmic reticulum stress, FOXO3a and p73 .
We determined p53 protein levels in HeLa and SiHa cells treated with PTX, ADR and PTX + ADR and its relationship with HPV E6 and E7 genes from HPV 18 and 16 respectively. Interestingly, ADR induced p53 protein stabilization in SiHa cells and reduced mRNA levels of HPV E6 and E7 in both cancer cells. In this sense, besides the fact that ADR did not modify the mRNA levels of p53 in SiHa cells, it impaired the degradation that could lead to growth arrest or apoptosis. If survival factors are also induced then growth arrest is favoured and the cell goes in senescence. Premature senescence has been described to be associated with increases in p53, p21, and p16 levels [35, 36].
When we measured the levels of mRNA expression of HPV E6 and E7 in PTX + ADR treated cells we observe a down-regulation in SiHa and an opposite effect in HeLa cells. In this respect we can only speculate that the transcriptional machinery used by HPV 16 and 18 is modulated differentially by PTX.
It has been described that up-regulation of p16 is associated with senescence [35, 36]. We detected up-regulation of p16 gene when SiHa and HeLa cells were treated with ADR and this is in agreement with the senescence observed in these cells and contrarily when the cells were treated with PTX.
Our observations show antitumor activity of PTX. This molecule induces apoptosis in a p53 independent manner and decreases the senescence induced by ADR. Importantly, PTX increases the sensitivity of cancer cells to the toxic effects of ADR by caspase participation and regulation of caspase -3, caspase-9, puma, noxa, and diablo genes.
HeLa (HPV 18+) and SiHa (HPV 16+) cervical cancer cell lines and the spontaneously immortalized human epithelial cell line HaCaT (used as non-tumorigenic control cells) were kindly provided by Dr. Boukamp (DKFZ-Heidelberg, Germany). All the cell lines were maintained in vitro and propagated in Dulbecco's modified Eagle's culture medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1× L-glutamine (2 mM final concentration) and antibiotics (penicillin/streptomycin), (this medium will be referred to as DMEM-S), and incubated at 37°C in an humidified atmosphere containing 95% air and 5% CO2. All the previously mentioned products were obtained from GIBCO™ Invitrogen Corporation (Carlsbad, CA, USA).
Drugs and experimental conditions
Adriamycin (ADR) was obtained from Lemery Laboratories, México, and stocked at 4°C less than 4 days and adjusted to desirable concentration with DMEM culture medium immediately before utilization. Pentoxifylline (PTX) (Sigma, Saint Louis MO, USA) dissolved in sterile saline solution 0.15 M, at a concentration of 0.2 M and kept at 4°C less than 4 days.
Cell culture and in vitro treatments
HeLa, SiHa and HaCaT cells suspended in DMEM-S at concentrations of 1.5 × 106 or 2 × 106/8 mL in exponential phase were seeded in p100 Petri dishes for flow cytometry assays, senescence and Western blot studies. For survival test and for apoptosis determined by ELISA, the cells were cultured in 96-well plates at a concentration of 3 × 105 cells/well/200 μL (final volume). For clonogenic assays the cells were seeded at densities of 1 × 104 cells/2 mL in 6-well plates. In all cases the cells were cultured overnight at 37°C in a humidified atmosphere containing 5% of CO2 and 95% air. The medium was then replaced with DMEM-S. Then the cells were either treated with PTX 8 mM, or ADR 1 μM or PTX + ADR (final concentrations). The cells were incubated with PTX 1 hour before ADR addition and 24 hours later the culture cells were harvested. In the cases for genes expression study the cells were incubated with the drugs for only 3 hours.
Cells survival was determined by the cleavage of tetrazolium salt WST-1 into formazan by cellular mitochondrial dehydrogenase enzyme. 24 hours after each treatment the cell were incubated as above indicated with 10 μL/well of WST-1/ECS solution (BioVision Research, Mountain View, CA, USA) and incubated for 3 hours in the same conditions. The absorbance (450 nm) of the treated and untreated samples was determined on a microtiter plate reader (Synergy™ HT Multi-Mode Microplate Reader. Biotek Winooski, VT, USA). Data are reported in percentage of cell survival as compared to respectively untreated control group considering as 100%.
Clonogenic Survival Assays
Cells were assayed for the cytotoxic effects of PTX or ADR or PTX + ADR after cell survival according to the established methods of performing the clonogenic assay . Subconfluent cultures were exposed to drugs for 6 hours. Then the cells were washed with PBS preheated to 37°C, trypsinized and plated in 6-well plates (100 cells/wells). After 12 days of incubation in complete culture medium, the colonies were stained with crystal violet after fixation with formaldehyde and counted manually. In each case the results are expressed as survival fraction which was obtained by dividing the number of colonies after the treatment by the number of cells seeding and the results multiply by the plate efficiency (PE). PE = (N°. of colonies formed/N° of cells seeded) ×100.
Adriamycin efflux assay
HeLa, SiHa and HaCaT cells were incubated with ADR for 1 hour in DMEM-S at 37°C. For ADR efflux measurement, the cells were washed with DMEM-S and treated or untreated with PTX for 2 hours at 37°C. The cells were washed, trypsinized and collected in drug-free medium. ADR fluorescence was determined in FACSAria cell sorter (BD, Bioscience, San Jose, CA, USA). For tests at least 20,000 events were analyzed for each sample. Data were processed with the FACSDiva software package (BD). The results are reported as the mean intensity fluorescence.
Apoptosis and caspase activity detection methods
Cellular detection of Annexin V, H2A.X phosphorylation and M30 caspase activity were determined by flow cytometry using the fluorescein isothiocyanate conjugated monoclonal Annexin V-FITC apoptosis Kit (Annexin-V-Fluos; Roche, Mannheim, Germany), H2A.X phosphorylation assay kit (H2A.X Phosphorylation Assay Kit, Millipore Temecula, CA, USA) and M30 CytoDEATH™ Biotin antibody (Roche Mannheim, Germany) respectively according to the manufacturer's instructions. For the three tests at least 20,000 events were analysed for each sample in an EPICS XL-MCL™ Flow Cytometer Beckman Coulter model (Fullerton, CA, USA). Data were processed with the System II software package (Beckman Coulter).
Apoptosis Detection by ELISA
In normal untreated and treated cell cultures we determinated cytoplasmic histone-associated-DNA-fragments (mono- and oligonucleosomes) spectrophotometrically (420 nm) using Cell Death Detection ElisaPLUS (Roche Mannheim, Germany) according the manufacturer's instruction . The enrichment of mono- and oligonucleosomes released into the cytoplasm was calculated: experimental absorbance/corresponding control absorbance. The results are expressed as the percentage of DNA fragmentation.
Acridine orange/ethidium bromide staining to detect late apoptosis by UV-microscopy
Briefly the cells were stained with ethidium bromide (Sigma Saint Louis MO, USA) and acridine orange (Sigma, Saint Louis MO, USA) (100 μg/mL each). Two hundred cells were counted and number of each of the following 4 cellular states recorded: i) live cells with normal nuclei (LN), bright green chromatin and organized structure; ii) apoptotic cells (A) with highly condensed or fragmented bright green-yellow chromatin; iii) dead cells with normal nuclei (DN), bright red chromatin and organized structure and iv) dead cells with apoptotic nuclei (DA) and bright orange chromatin, which were highly condensed and fragmented. Apoptotic index (AI): A + DA/LN +A +DN +DA × 100.
β-galactosidase associated senescence
According to the manufacturer's instructions senescence was determined by histochemical in treated and untreated control cells by Senescence Detection Kit (BioVision Mountain View, CA, USA) which detects β-galactosidase activity (SA-β-gal) present in senescence cells. We counted 300 cells of six microscopic fields to determine the percentage of SA-β-gal stained positive cells identified by an intense blue stain in the membrane.
Protein extraction for IκBα ELISA and p53 western blot assay
1-2 × 106 cells were seeded in p100 culture Petri-dishes and treated next day with PTX, ADR and PTX + ADR for 24 hours. After treatment, cells were harvested by scraping and lysed with RIPA buffer (0.5% deoxycholate, 0.5% NP-40, 0.5% SDS, 50 mM Tris pH 7.4 and 100 mM NaCl) containing protein inhibitors. Following sonication (15 pulses, 50% amp), protein extracts were obtained after 30 min incubation at 4°C and 5 min centrifugation at 14 000 rpm/4°C. Protein concentrations were determined using BioRad Dc Protein Kit.
The levels of IκBα protein was determined in HeLa, SiHa and HaCaT treated and untreated control cells using a commercial ELISA kit at 450 nm (Invitrogen), according to the manufacturer's instructions. The results are expressed in pg/mL.
p53 Western blot analyses
Total cell protein (50 μg) was subjected to electrophoresis using a 12% sodium dodecyl sulfate polyacrylamide gel. Subsequently, proteins were transferred to Immobilon-P PVDF membranes (Millipore) and incubated with 1× western blocking reagent (Roche) during 1 hour for nonspecific binding. Immunodetection of p53 was performed using anti-p53 (DO-1, dilution 1:750, Santa Cruz biotechnology, Inc.) at room temperature for 2 hours. After incubation with a horseradish peroxidase-conjugated secondary antibody, immunoreactive proteins were visualized by western blotting luminol reagent in chemiluminiscence films and resolved on BioMax film (Kodak) according to the manufacture's specifications. Control β-actin antibody.
Quantitative real time PCR
Primer pairs used for real-time quantitative PCR
Primer pair sequences
5'CTC CGG AGG ATG AGT GAC GAGT 3'
5'ACT TCC GCC CAT ATT CAA GAT 3'
5'CGC TTC GTG GTC GAC TTC AT 3'
5'AGA AGG CAA AGA CTT CGC TTA 3'
5'TTT GCT TCA GGG TTT CAT CC 3'
5'CAG TTG AAG TTG CCG TCA GA 3'
5'GAG ATG CCT GGG AAG AAG G 3'
5'TCC TGA GCA GAA GAG TTT GGA 3'
5' GAT GGC GGA CGA CCT CAA C 3'
5'TGG GAG TCC AGT ATG CTA CAT GGT 3'
5'TGA GCT GCA GGT TCC TTA TCT G 3'
5'GAA TGG CTT TGT GCT TAG TTT T 3'
5'TGA CTT CAA AAC ACC AAG AGT A 3'
5'TTT CTG ACG GAG CTC TTC TA 3'
5'CGA CTT TGT CAC CGA GAC AC 3'
5'CGT TTT CGA CCC TGA GAG T 3'
5'CTG AGG TTG GCT CTG ACT GTA CCA CCA TCC 3'
5'CTC ATT CAG CTC TCG GAA CAT CTC GAA GCG 3'
5'CAG TAA CCA TGC CCG CAT AGA T 3'
5'TGA AAA GGC AGA AGC GGT GT 3'
5'CAC GAG ACG GTC TTC CAA GGA TGC T 3'
5'CTA GGT TGC TAG GGT GCA ACT CTA GGA 3'
5'GCA GGC GAC GAG TTT GAA CT 3'
5'GTG TCT GGT CAT TTC CGA CTG A 3'
5'ATA CTC CAC AGC ACC TGG TTA T 3'
5'AAT GAG AGG GAA ATA CAG TAC CAA 3'
5'GTA CGT TGA GAC CCT GGA CGA C 3'
5'GCT GCT AAG AGC CTG TCT GTC ACT 3'
E6 (HPV 18)
5'GCG ACC CTA CAA GCT ACC TGA T 3'
5'GCA CCG CAG GCA CCT TAT TA 3'
E7 (HPV 18)
5'TGT CAC GAG CAA TTA AGC GAC T 3'
5'CAC ACAAAG GAC AGG GTG TTC A 3'
E6 (HPV 16)
5'CAG AGC TGC AAA CAA CTA TAC 3'
5'AGT GGC TTT TGA CAG TTA ATA C3
E7 (HPV 16)
5'GAC AAG CAG AAC CGG ACA G 3'
5'ATT CCT AGT GTG CCC ATT AAC A 3'
5'GCA TTG ACA ACA GGG TTC GTA G 3'
5'ATT TAA ACA GAA AAC GTG CAC A 3'
Results of each experiment represent mean ± standard deviation (SD) of 3 independent experiments carried out in triplicate. Differences between groups were determined by Student "t" test and ANOVA. Significant results were considered with p < 0.05. For the comparison of gene expression, it was only considered positive or negative differences ≥30% in relationship to reference values. In some cases was calculated the Δ% that represent the percent of increment or diminution in relationship to comparative group.
The authors are grateful to Professor Orbach-Arbouys Simone (Service De Pharmacologie, Hôpital Paul Brouse. Villejuif, France) for helpful discussion and critical reading of the manuscript. We finally thank to our qualified staff Leticia Ramos-Zavala and Maria de Jesus Delgado-Avila.
- Arreygue-Garcia NA, Daneri-Navarro A, del Toro-Arreola A, Cid-Arregui A, Gonzalez-Ramella O, Jave-Suarez LF, Aguilar-Lemarroy A, Troyo-Sanroman R, Bravo-Cuellar A, Delgado-Rizo V, Garcia-Iglesias T, Hernandez-Flores G, Del Toro-Arreola S: Augmented serum level of major histocompatibility complex class I-related chain A (MICA) protein and reduced NKG2D expression on NK and T cells in patients with cervical cancer and precursor lesions. BMC Cancer. 2008, 8: 16- 10.1186/1471-2407-8-16PubMed CentralView ArticlePubMedGoogle Scholar
- Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ: Cancer statistics. CA Cancer J Clin. 2008, 58: 71-96. 10.3322/CA.2007.0010View ArticlePubMedGoogle Scholar
- Alvarez-Salas LM, DiPaolo JA: Molecular approaches to cervical cancer therapy. Curr Drug Discov Technol. 2007, 4: 208-219. 10.2174/157016307782109661View ArticlePubMedGoogle Scholar
- Dominguez-Rodriguez JR, Gomez-Contreras PC, Hernandez-Flores G, Lerma-Diaz JM, Carranco A, Cervantes-Munguia R, Orbach-Arbouys S, Bravo-Cuellar A: In vivo inhibition by antioxidants of adriamycin-induced apoptosis in murine peritoneal macrophages. Anticancer Res. 2001, 21: 1869-1872.PubMedGoogle Scholar
- Herr I, Debatin KM: Cellular stress response and apoptosis in cancer therapy. Blood. 2001, 98: 2603-2614. 10.1182/blood.V98.9.2603View ArticlePubMedGoogle Scholar
- Shabbits JA, Hu Y, Mayer LD: Tumor chemosensitization strategies based on apoptosis manipulations. Mol Cancer Ther. 2003, 2: 805-813.PubMedGoogle Scholar
- Dominguez-Rodriguez JR, Chaparro-Huerta V, Hernandez-Flores G, Gomez-Contreras PC, Lerma-Diaz JM, Ortiz-Lazareno PC, Cervantes-Munguia R, Orbach-Arbouys S, Scott-Algara D, Bravo-Cuellar A: In vivo adriamycin-induced apoptosis in peritoneal murine macrophages: partial participation of a caspase cascade. Anticancer Res. 2004, 24: 2689-2696.PubMedGoogle Scholar
- Elmore S: Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007, 35: 495-516. 10.1080/01926230701320337PubMed CentralView ArticlePubMedGoogle Scholar
- Hayflick L: The Limited in Vitro Lifetime of Human Diploid Cell Strains. Exp Cell Res. 1965, 37: 614-636. 10.1016/0014-4827(65)90211-9View ArticlePubMedGoogle Scholar
- Roninson IB: Tumor cell senescence in cancer treatment. Cancer Res. 2003, 63: 2705-2715.PubMedGoogle Scholar
- Petignat P, Roy M: Diagnosis and management of cervical cancer. Bmj. 2007, 335: 765-768. 10.1136/bmj.39337.615197.80PubMed CentralView ArticlePubMedGoogle Scholar
- Elmore LW, Rehder CW, Di X, McChesney PA, Jackson-Cook CK, Gewirtz DA, Holt SE: Adriamycin-induced senescence in breast tumor cells involves functional p53 and telomere dysfunction. J Biol Chem. 2002, 277: 35509-35515. 10.1074/jbc.M205477200View ArticlePubMedGoogle Scholar
- Vigneron A, Roninson IB, Gamelin E, Coqueret O: Src inhibits adriamycin-induced senescence and G2 checkpoint arrest by blocking the induction of p21waf1. Cancer Res. 2005, 65: 8927-8935. 10.1158/0008-5472.CAN-05-0461View ArticlePubMedGoogle Scholar
- Hornsby PJ: Senescence as an anticancer mechanism. J Clin Oncol. 2007, 25: 1852-1857. 10.1200/JCO.2006.10.3101View ArticlePubMedGoogle Scholar
- Park S, Hatanpaa KJ, Xie Y, Mickey BE, Madden CJ, Raisanen JM, Ramnarain DB, Xiao G, Saha D, Boothman DA, Zhao D, Bachoo RM, Pieper RO, Habib AA: The receptor interacting protein 1 inhibits p53 induction through NF-kappaB activation and confers a worse prognosis in glioblastoma. Cancer Res. 2009, 69: 2809-2816. 10.1158/0008-5472.CAN-08-4079PubMed CentralView ArticlePubMedGoogle Scholar
- Nakanishi C, Toi M: Nuclear factor-kappaB inhibitors as sensitizers to anticancer drugs. Nat Rev Cancer. 2005, 5: 297-309. 10.1038/nrc1588View ArticlePubMedGoogle Scholar
- Wang CY, Cusack JC, Liu R, Baldwin AS: Control of inducible chemoresistance: enhanced anti-tumor therapy through increased apoptosis by inhibition of NF-kappaB. Nat Med. 1999, 5: 412-417. 10.1038/10577View ArticlePubMedGoogle Scholar
- Cusack JC, Liu R, Baldwin AS: NF- kappa B and chemoresistance: potentiation of cancer drugs via inhibition of NF- kappa B. Drug Resist Updat. 1999, 2: 271-273. 10.1054/drup.1999.0094View ArticlePubMedGoogle Scholar
- Bentires-Alj M, Merville MP, Bours V: NF- kappa B and chemoresistance: could NF- kappa B be an antitumor target?. Drug Resist Updat. 1999, 2: 274-276. 10.1054/drup.1999.0093View ArticlePubMedGoogle Scholar
- Bravo-Cuellar A, Gomez-Contreras PC, Lerma-Diaz JM, Hernandez-Flores G, Dominguez-Rodriguez JR, Ortiz-Lazareno P, Cervantes-Munguia R, Orbach-Arbouys S: In vivo modification of adriamycin-induced apoptosis in L-5178Y lymphoma cell-bearing mice by (+)-alpha-tocopherol and superoxide dismutase. Cancer Lett. 2005, 229: 59-65. 10.1016/j.canlet.2005.01.013View ArticlePubMedGoogle Scholar
- Lerma-Diaz JM, Hernandez-Flores G, Dominguez-Rodriguez JR, Ortiz-Lazareno PC, Gomez-Contreras P, Cervantes-Munguia R, Scott-Algara D, Aguilar-Lemarroy A, Jave-Suarez LF, Bravo-Cuellar A: In vivo and in vitro sensitization of leukemic cells to adriamycin-induced apoptosis by pentoxifylline. Involvement of caspase cascades and IkappaBalpha phosphorylation. Immunol Lett. 2006, 103: 149-158. 10.1016/j.imlet.2005.10.019View ArticlePubMedGoogle Scholar
- Bohm L, Roos WP, Serafin AM: Inhibition of DNA repair by Pentoxifylline and related methylxanthine derivatives. Toxicology. 2003, 193: 153-160. 10.1016/S0300-483X(03)00294-4View ArticlePubMedGoogle Scholar
- Lee SJ, Cho YS, Cho MC, Shim JH, Lee KA, Ko KK, Choe YK, Park SN, Hoshino T, Kim S, Dinarello CA, Yoon DY: Both E6 and E7 oncoproteins of human papillomavirus 16 inhibit IL-18-induced IFN-gamma production in human peripheral blood mononuclear and NK cells. J Immunol. 2001, 167: 497-504.View ArticlePubMedGoogle Scholar
- Gomez-Contreras PC, Hernandez-Flores G, Ortiz-Lazareno PC, Del Toro-Arreola S, Delgado-Rizo V, Lerma-Diaz JM, Barba-Barajas M, Dominguez-Rodriguez JR, Bravo-Cuellar A: In vitro induction of apoptosis in U937 cells by perillyl alcohol with sensitization by pentoxifylline: increased BCL-2 and BAX protein expression. Chemotherapy. 2006, 52: 308-315. 10.1159/000096003View ArticlePubMedGoogle Scholar
- Meissner JD: Nucleotide sequences and further characterization of human papillomavirus DNA present in the CaSki, SiHa and HeLa cervical carcinoma cell lines. J Gen Virol. 1999, 80 (7): 1725-1733.View ArticlePubMedGoogle Scholar
- Rishi L, Gahlot S, Kathania M, Majumdar S: Pentoxifylline induces apoptosis in vitro in cutaneous T cell lymphoma (HuT-78) and enhances FasL mediated killing by upregulating Fas expression. Biochem Pharmacol. 2009, 77: 30-45. 10.1016/j.bcp.2008.09.018View ArticlePubMedGoogle Scholar
- Braig M, Schmitt CA: Oncogene-induced senescence: putting the brakes on tumor development. Cancer Res. 2006, 66: 2881-2884. 10.1158/0008-5472.CAN-05-4006View ArticlePubMedGoogle Scholar
- Krtolica A, Parrinello S, Lockett S, Desprez PY, Campisi J: Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging. Proc Natl Acad Sci USA. 2001, 98: 12072-12077. 10.1073/pnas.211053698PubMed CentralView ArticlePubMedGoogle Scholar
- Campisi J: Cellular senescence as a tumor-suppressor mechanism. Trends Cell Biol. 2001, 11: S27-S31.View ArticlePubMedGoogle Scholar
- Drobna Z, Stein U, Walther W, Barancik M, Breier A: Pentoxifylline influences drug transport activity of P-glycoprotein and decreases mdrl gene expression in multidrug resistant mouse leukemic L1210/VCR cells. Gen Physiol Biophys. 2002, 21: 103-109.PubMedGoogle Scholar
- Serafin AM, Binder AB, Bohm L: Chemosensitivity of prostatic tumour cell lines under conditions of G2 block abrogation. Urol Res. 2001, 29: 221-227. 10.1007/s002400100186View ArticlePubMedGoogle Scholar
- Shiu RP, Newsham IF, Gewirtz DA: Apoptosis, autophagy, accelerated senescence and reactive oxygen in the response of human breast tumor cells to Adriamycin. Biochem Pharmacol. 2009, 77: 1139-1150. 10.1016/j.bcp.2008.12.016View ArticlePubMedGoogle Scholar
- Horvath B, Vekasi J, Kesmarky G, Toth K: In vitro antioxidant properties of pentoxifylline and vinpocetine in a rheological model. Clin Hemorheol Microcirc. 2008, 40: 165-166.PubMedGoogle Scholar
- Li J, Lee B, Lee AS: Endoplasmic reticulum stress-induced apoptosis: multiple pathways and activation of p53-up-regulated modulator of apoptosis (PUMA) and NOXA by p53. J Biol Chem. 2006, 281: 7260-7270. 10.1074/jbc.M509868200View ArticlePubMedGoogle Scholar
- Takahashi A, Ohtani N, Yamakoshi K, Iida S, Tahara H, Nakayama K, Nakayama KI, Ide T, Saya H, Hara E: Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence. Nat Cell Biol. 2006, 8: 1291-1297. 10.1038/ncb1491View ArticlePubMedGoogle Scholar
- Lehmann BD, Paine MS, Brooks AM, McCubrey JA, Renegar RH, Wang R, Terrian DM: Senescence-associated exosome release from human prostate cancer cells. Cancer Res. 2008, 68: 7864-7871. 10.1158/0008-5472.CAN-07-6538View ArticlePubMedGoogle Scholar
- Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C: Clonogenic assay of cells in vitro. Nat Protoc. 2006, 1: 2315-2319. 10.1038/nprot.2006.339View ArticlePubMedGoogle Scholar
- Aguilar-Lemarroy A, Gariglio P, Whitaker NJ, Eichhorst ST, zur Hausen H, Krammer PH, Rosl F: Restoration of p53 expression sensitizes human papillomavirus type 16 immortalized human keratinocytes to CD95-mediated apoptosis. Oncogene. 2002, 21: 165-175. 10.1038/sj.onc.1204979View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.