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p-Akt signaling on HeLa cells
Biomedicine & Pharmacotherapy 82 (2016) 124–132
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Interferon b improves the efficacy of low dose cisplatin by inhibiting NF-kB/p-Akt signaling on HeLa cells Purushoth Ethiraja , Karpagam Veerappanb , Shila Samuelb , Sundaresan Sivapathama,* a b
Department of Medical Research, SRM Medical College Hospital and Research Centre, SRM University, Kattankulathur 603203, Tamilnadu, India Department of Biochemistry, VRR Institute of Biomedical Science (Affiliated to University of Madras), Chennai 600 056, India
A R T I C L E I N F O
Article history: Received 19 January 2016 Received in revised form 26 April 2016 Accepted 26 April 2016 Keywords: Cisplatin Interferon b Chemosensitivity NF-kB Akt PARP
A B S T R A C T
The purpose of this study was to evaluate the anticancer efficacy of interferon b in combination with low dose of cisplatin on human cervical cancer progression, as well as its principal action mechanism. The combination treatment synergistically potentiated the effect of interferon b on cell growth inhibition and DNA damage on HeLa cells by repressing NF-kB/p-Akt signaling. Synergistic targeting of these pathways has a therapeutic potential. Further, the combination treatment ameliorated the expression of proapoptotic Bax, and decreased the expression of anti-apoptotic protein Bcl-2. Additionally, the expression of active PARP was significantly increased and MMP-9 level was decreased in combination group as compared to the expression seen for the treatment with interferon b or cisplatin alone. Results demonstrate that the synergistic inhibitory effects of interferon b and low dose of cisplatin on human cervical cancer cells and also suggest that the inhibition of NF-kB/p-Akt signaling pathway plays a critical role in the anticancer effects of combination treatment along with the induction of PARP. Therefore, the combination of interferon b and cisplatin may be a useful treatment for human cervical cancer, with a greater effectiveness than other treatments. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Cervical cancer is the second most frequent cause of cancer in women [1]. Cisplatin is an effective agent in its treatment [2]. However, its usage is limited by its toxicity and acquired chemoresistance through the course of treatment [3]. Barr et al. stated that increased doses of cisplatin treatment for long term on lung cancer cell lines, led to cell lines resistant to cisplatin mediated cell death [4]. Activation of p-Akt and the NF-kB/Bcl-2 pathways leads to inhibition of chemotherapy-induced apoptosis, which results in treatment resistance [5,6]. Activation of NF-kB has been identified as a key mechanism of cisplatin resistance. NF-kB activity is inversely correlated with cellular sensitivity to chemotherapy in carcinoma cell lines [7]. NF-kB promotes the migration and metastasis of hepatocellular carcinoma cells [8], cervical cancer cells [9], and breast cancer cells [10] through an upregulation of Matrix Metalloproteinases (MMPs). Suppression of NF-kB activation is effective in the prevention and treatment of cancer [6]. Li and Sethi reported that chemoresistance is mediated
* Corresponding author. E-mail address: [email protected] (S. Sivapatham). http://dx.doi.org/10.1016/j.biopha.2016.04.058 0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.
through several genes regulated by NF-kB and inhibition of this transcription factor increases sensitivity of cancer cells to the apoptotic action of chemotherapeutic agents [11]. Thus, it was hypothesised that agents inhibiting the activation of NF-kB may exhibit the therapeutic potential for the suppression of carcinogenesis and tumor metastasis [12]. The antitumor effects of interferon b (IFN-b) have been attributed to the inhibition of cell proliferation, induction of cell differentiation, immunomodulation and alteration of the level of gene expression in target cells [13]. The binding of IFN-b to the type I IFN receptor results in the phosphorylation and activation of tyrosine kinase2 (Tyk2) and Janus kinase1 (JAK1), which in turn regulates the phosphorylation and activation of signal transducers and activators of transcription (STAT) [14]. Josiane et al. reported that IFN-induced IRF1 (interferon regulatory factor-1) inhibited NF-kB dependent activation of MMP-9 by binding to the promoter region overlapping the NF-kB binding site [15]. Li et al. demonstrated that IFN-b produced synergistic anti-tumour effects with cisplatin through up-regulated p53 expression on mesothelioma cells [16]. Hence to improve the sensitivity and efficacy of cisplatin at lower doses it is combined with IFN-b to achieve synergistic effect. We previously reported the synergistic anti-carcinogenic effect of IFN-b with cisplatin on human breast adenocarcinoma
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MDA MB231 cells [17]. In this study, we aimed to evaluate the effect of IFN-b individually and in combination with low of dose cisplatin on cellular proliferation, genotoxicity, and apoptosis in HeLa cells. 2. Materials and methods 2.1. Drugs and reagents Interferon b Human (Cat No.I4151, Purity 95%), platinum(II) (Cat No.479306), cytochalasin B (Cat No. C6762), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Cat No. M2003) and dimethyl sulphoxide (DMSO) (Cat No.D2438) were purchased from Sigma Aldrich (Sigma Aldrich Co., USA). Antibodies against STAT2, p-STAT2 (Tyr690), Bax, Bcl-2, p-Akt (Ser473), NF-kB (p65), PARP and MMP9 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against IkB and p-IkB were purchased from Cell Signaling Technology, Inc (MA, USA). Horseradish peroxidase conjugated anti-mouse was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2.2. Maintenance of cell lines The cell lines HeLa obtained from National Centre for Cell Science (Pune, India) were maintained and grown in a humidified incubator at 37 C with 5% CO2. Cells were grown as a monolayer in plastic tissue culture flasks in Dulbecco’s Modified Eagles Medium (DMEM) (GIBCO, Grand Island, New York, USA). The media was supplemented with 10% fetal bovine serum (FBS) (GIBCO, Grand Island, New York, USA) and antibiotics (Penicillin 50 IU/mL, Streptomycin 3.5 mg/mL and Gentamycin 2.5 mg/mL) (GIBCO, Grand Island, New York, USA).
2.3. Cell proliferation assay HeLa cells were seeded in 96-well plates at a density of 5 103 cells/well in 200 mL DMEM containing 10% FBS and incubated overnight. Nonadherent cells were removed by gentle washing after 24 h. Then cells were replaced with serum free medium with varying concentrations of interferon b (100–2500 IU/mL) and cisplatin (0.01–100 mM). A negative control containing serum free medium with DMSO was also evaluated. After 72 h of treatment, the plates were incubated with 20 mL 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) solutions (5 mg/mL) for 3 h at 37 C. The formazan was dissolved in 150 mL/well dimethyl sulfoxide (DMSO) and the absorbance was detected at 590 nm using microplate reader (Bio Rad, USA). Cell viability was expressed as a percentage of untreated cells, which served as the negative control group and was designated as 100%; the results were expressed as a percentage of the negative control. The median inhibitory concentration (IC50) (defined as the drug concentration at which cell growth was inhibited by 50%) was assessed from the dose response curves.
2.4. Synergistic analysis The synergistic effect of IFN-b with cisplatin was examined using median effect method as originally described by Chou and Talalay [18]. The combination index (CI) values reflect the nature of the interaction between IFN-b and cisplatin. The CI < 1 indicates synergy, CI = 1, addictive, CI > 1, antagonism. The combination index analysis was performed using Compusyn software (comboSyn, Inc., Paramus, (NJ)).
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2.5. Tryphan blue dye exclusion assay HeLa cells were treated with IFN-b and cisplatin at 1500 IU/mL and 12 mM, respectively. The low dosage of cisplatin (6 mM) was also studied. For the combination, IFN-b (1500 IU/mL) and cisplatin (6 mM half of the IC50) were treated for 72 h. Cell viability was determined by the tryphan blue dye exclusion assay; this dye excluded living cells and only penetrated the cell membrane of dead cells. The proportion of dead cells was determined by counting the number of stained cells using a hemocytometer. 2.6. Western blotting The cells were seeded in 24-well plates at a density of 5 104 cells/well in 1 mL of DMEM containing 10% FBS overnight. Nonadherent cells were removed by gentle washing after 24 h. Then, cells were treated with interferon b (1000 & 1500 IU/mL) for STAT and p-STAT2 (Tyr690). The cells were treated with interferon b and cisplatin at 1500 IU/mL and 12 mM respectively, for combination, 1500 IU/mL of IFN-b and half of the obtained concentration of IC50 (6 mM) of cisplatin were used for t-Akt, p-Akt (Ser473), IkB, pIkB, NF-kB (p65), Bax, Bcl-2, PARP, MMP-9. A negative control containing serum free medium was also evaluated. After 48 h of treatment, cells were lysed by the addition of cold RIPA buffer [150 mM NaCl, 50 mM Tris HCL, 0.1% SDS, 1% Triton X-100, 1 mM PMSF, 2 mM NaF, Na3VO4, b-glycerophosphate and 2 mM EDTA, and fresh protease inhibitor cocktail (Cat No. P8340, Sigma Aldrich)] and cell lysate was centrifuged at 14,000 rpm at 4 C for 20 min. The supernatant was harvested and analysed for protein content using BCA method (Cat No. 23227, Pierce, USA). Protein was denatured in sample buffer, then separated on 12% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (semidry trans-blot system). The blots were blocked for 2 h at room temperature with Tris-Buffered Saline (TBS, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 5% non-fat milk. The blots were washed three times with TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.02% Tween 20) and incubated with specific primary antibodies (1:1000 dilutions) at 4 C overnight. The blots were incubated for 1 h at room temperature with secondary antibodies (1:5000 dilutions), and detected by ECL detection reagent. To ensure that equal amounts of sample protein were applied for electrophoresis, b-actin was used as an internal control. Densitometric analysis was done using ImageJ software. 2.7. Analysis of DNA damage using micronucleus assay The cytotoxicity test (MTT) demonstrated that IFN-b and cisplatin at 1500 IU/mL and 12 mM respectively produced 50% reduction in cell viability (IC50). These concentrations were used for micronuclei (MN) analysis. For combination, 1500 IU/mL of IFNb and half of the obtained IC50 concentration (6 mM) of cisplatin were used. The MN assay was performed using a slight modification of previously published method of Fenech [19]. Cells were seeded at 1 105 in 60 mm dishes. After 24 h, nonadherant cells were removed by gentle washing with PBS and then cells were treated with concentrations as mentioned above, alone and in combination. Cytochalasin-B (3 mg/mL) was added at 44th h to arrest cells at the cytokinesis stage. The cells were harvested after 72 h. The cells were subjected to a mild treatment with hypotonic solution (0.45% KCl). Then, the cells were centrifuged (1000 rpm, 10 min) and fixed three times in cold methanol and acetic acid (3:1). The initial addition of fixative contained 1% formaldehyde, which enhanced cytoplasm preservation. The cells were dropped on cold glass slides, stained with 10% Giemsa solution (pH 6.8, GIBCO, Burlington, USA) for 5 min. The slides were coded and scored
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under a light microscopy at 400* magnification or 1000* as required. Thousand binucleated cells (cells with two daughter nuclei surrounded by cytoplasm) were scored for the presence of MN according to the scoring criteria given by Fenech [19]. Based on the recorded data, the frequency of micronuclei (MNi) was calculated using the formula MNi = a/b and the error was calculated by p MNi = a/b where ‘a’ is the total number of micronuclei and ‘b’ is the total number (1000) of binucleated cells scored. The ability of the cells to proliferate in vitro was evaluated by counting the number of cells with one, two and four MN on the same slide using the nuclear division index (NDI), calculated
according to the formula: NDI = (MI + 2MII + 3MIII + 4MIV)/N, where MI to MIV represent the number of cells with one to four nuclei, respectively, and N is the number of cells scored [19].
2.8. Statistical analysis Data are presented as Mean SEM. Each value is the mean of at least three separate experiments. Statistical evaluation was performed using an unpaired Student’s t-test. p values of <0.05 was considered to be statistically significant.
Fig. 1. HeLa cells were treated with cisplatin and IFN-b cell proliferation was assessed using the MTT assay. Cells were treated with various concentrations of Cisplatin (0.01– 100 mM) (A) Interferon b (100–2500 IU/ml) (B) and combination of cisplatin (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 100) with IFN-b (1500 IU/ml) (C). Cell proliferation was determined by MTT. Data represented are Mean SEM (n = 3).
P. Ethiraj et al. / Biomedicine & Pharmacotherapy 82 (2016) 124–132 Table 1 Synergistic combinatory effect of cisplatin with IFN-b. Cells were treated with varying concentrations of cisplatin (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100) + IFN-b (1500 IU/ml). CI values between 0.1–0.3 shows strong synergism. Cisplatin (mM)
IFN-b (IU/ml)
CI Values
0.01 0.05 0.1 0.5 1 5 10 50 100
1500 1500 1500 1500 1500 1500 1500 1500 1500
0.784 0.721 0.681 0.592 0.351 0.331 0.281 0.246 0.212
CI indicates combination index.
3. Results 3.1. Inhibition of growth in HeLa cells by IFN-b and its synergistic effect with anticancer agent cisplatin The effects of IFN-b and cisplatin on the proliferation of HeLa cells were examined. The percentage of viable cells obtained in the MTT assay with varying concentrations of
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cisplatin (0.01–100 mM) and IFN-b (100–2500 IU/mL) were shown in Fig. 1(A and B). Dramatic dose-dependent reduction on the cell proliferation was observed in both IFN-b and cisplatin treated cells after 72 h of exposure. The IC50 values of interferon b and cisplatin were 1500 IU/mL and 12 mM respectively. Synergistic effect (CI < 1) on anti-proliferation was observed when different concentrations of cisplatin was combined with 1500 IU/mL of IFN-b in Table 1. 3.2. IFN-b induced p-STAT2 (Try690) signaling required for inhibition of cell proliferation Fig. 2(A) showed the protein expression IFN-b induced STAT2 and p-STAT2 (Try690) signaling mediated cell proliferation inhibition on cells treated with 1000 and 1500 IU/mL. Densitometric analysis showed a significant difference in the expression of p-STAT2 in cells treated with 1500 IU/mL of IFN-b compared to control (Fig. 2(B)). 3.3. Effects of cisplatin/IFN-b and its synergistic combination on HeLa cell death To investigate the effects of IFN-b and/or cisplatin on survival or growth, HeLa cells were exposed with 1500 IU/mL of IFN-b,
Fig. 2. Protein expression of STAT2 and p-STAT2 (Try690) on cells treated with IFN-b. Hela cells were treated with 1000 and 1500 IU/mL (Dose of IC50) of IFN-b, whereas untreated cells were used as control (0), after 48 h protein expression of STAT2 and p-STAT2 was analysed using western blot. (A) Representative image of blot. (B) Densitometric analysis of p-STAT2 was normalized with STAT2 and represented as ratio (p-STAT2/STAT2), it showed significant difference compared to control. P < 0.05 (*) indicated significant difference, whereas p < 0.01 (**) showed more significance. The results are given as Mean SEM from three experiments. b-actin was used as an internal control.
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Fig. 3. Cell death caused by cisplatin and/or IFN-b treatment on HeLa cells. Percentage of cell death was significantly increased P < 0.05 (*) in cells treated with IFN-b and cisplatin (6 mM), whereas its shows more significant difference P < 0.01 (**) at cisplatin (12 mM), and highly significant p < 0.001 (***) increase at combination (Cisplatin (6 mM) + IFN-b (1500 IU/mL)) group when compared to control.
Fig. 4. Frequency of Micronuclei. The binucleated cells with micronucleus found after treatment of the predicted dose of IFN-b, cisplatin and its combination (Cisplatin (6 mM) + IFN-b (1500 IU/mL)) showed a significant difference, when compared to control. P < 0.05 (*) indicates significant difference, p < 0.01 (**) indicates more significant, whereas p < 0.001 (***) indicates highly significant difference. NS indicates Non-significant difference.
cisplatin at 6 and 12 mM and its combination for 72 h. Fig. 3 showed that the percentage of cell death was significantly increased (P < 0.05) in cells treated with IFN-b at 1500 IU/mL and cisplatin at 6 mM respectively, whereas more significant difference (P < 0.01) was observed in cells treated with cisplatin (12 mM) when compared to control. Highly significant (p < 0.001) cell death was observed at combination group, when compared to control.
3.5. NDI Table 2 represents NDI of control and treated cells. The cells treated with IFN-b and cisplatin alone showed a significant difference (p < 0.05) when compared to control. The NDI value of the combination group was 1.032 0.07 more significant (p < 0.01), compared to control.
3.4. Induction of micronuclei frequency on cells treated with IFN-b and cisplatin alone and in combination Fig. 4 showed the graphical representation of the micronuclei frequencies. Baseline micronuclei frequency obtained in untreated control cells was 0.018 0.004. There was no significant difference between vehicle control groups (DMSO) and control groups. IFN-b treated cells induced significant increase in MN frequencies when compared to control. Lower dosage of cisplatin (6 mM) induced MN frequencies were more significant (p < 0.01) compared to control. The micronuclei frequencies obtained in the cisplatin (12 mM) treated group was (0.0919 0.98) highly significant (p < 0.001), compared to control. The combinational group also showed highly significant difference among MN frequencies, when compared to control. There was no significant change when cisplatin group (12 mM) compared with combination group (6 mM cisplatin + 1500 IU/mL IFN-b).
Table 2 Illustrates obtained nuclear division index (NDI) values. Mean and standard deviation of nuclear division index (NDI) for 1500 cells analysed after treatment of HeLa cells to the predicted dose of IFN-b and cisplatin showed a significant difference P < 0.05 (*) when compared to control, whereas the combination (Cisplatin (6 mM) + IFNb (1500 IU/mL)) group showed more significant p < 0.01 (**). Control DMSO IFN-b Cisplatin (6 mM) Cisplatin (12 mM) Combination * **
p < 0.05. p < 0.01.
1.5122 0.11 1.516 0.12 1.272 0.08* 1.202 0.09* 1.120 0.10* 1.032 0.07**
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Fig. 5. Protein expression of Akt, p-Akt (Ser473), IkB, p- IkB, NF-kB on cells treated with cisplatin, IFN-b and its combination after 48 h was analysed using western blot. (A) Image of blot. (B) Densitometric analysis of p-Akt was normalized to t-Akt and represented as ratio (p-Akt/Akt), it showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P < 0.001 (***) indicated highly significant. (C) Densitometric analysis of p-IkB was normalized to t-IkB and represented as ratio (p-IkB/IkB), it showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P < 0.001 (***) indicated highly significant. (D) Densitometric analysis of NF-kB showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P < 0.001 (***) indicated highly significant. NS indicated non-significant difference. The results are given as Mean SEM from three experiments. b-actin was used as an internal control.
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3.6. IFN-b prevents cisplatin induced up-regulation of p-Akt(Ser473) in cells
and IFN-b displayed highly significant suppression on the expression of MMP-9 compared to control.
The protein expression of Akt and p-Akt (Ser473) on treated cells were studied. Fig. 5(A) exhibited the blot expression of Akt and p-Akt. Fig. 5(B) densitometric analysis exhibited cells treated with cisplatin (12 mM) showed non-significant change when compared to control. Cells treated with IFN-b and cisplatin (6 mM) showed more significant (p < 0.01) and significant (p < 0.05) down regulation in p-Akt respectively, when compared to control. The expression of p-Akt exhibited highly significant (p < 0.001) at combination group when compared to control.
4. Discussion
3.7. The diminished expression of p-IkB at combined treatment was observed in cells The protein expression of IkB and p-IkB on treated cells was studied. Fig. 5(A) exhibited the protein expression of IkB and p-IkB. Fig. 5(C) Densitometric analysis exhibited cells treated with cisplatin (12 mM) showed non-significant change when compared to control. Cells treated with IFN-b and cisplatin (6 mM) showed more significant (p < 0.01) down regulation in p-IkB when compared to control. The reduced expression level of p-IkB was significantly (p < 0.001) high at combination group when compared to control. 3.8. Inhibition of NF-kB (p65) expression was observed in cells treated with low dose of cisplatin with IFN-b The protein expression of NF-kB (p65) on treated cells was studied. Fig. 5(A) exhibited the protein expression of p65. Fig. 5(D) Densitometric analysis revealed cells treated with cisplatin (12 mM) showed non-significant change when compared to control. Cells treated with IFN-b and cisplatin (6 mM) showed significant (p < 0.05) and more significant (p < 0.01) down regulation in p65 when compared to control, respectively. The expression level of p65 was highly significant (p < 0.001) at reduced dosage of cisplatin (6 mM) when compared to control. 3.9. IFN-b and low dose of cisplatin sensitizes cervical cancer cells through Bcl-2 family members and induced the activity of PARP Fig. 6(A) showed the expression of Bcl-2 and Bax by immunoblotting. Fig. 6(B and C) densitometric analysis showed that IFN-b and low dose of cisplatin (6 mM) is able to markedly down-regulate the expression of Bcl-2 and increase the expression Bax in HeLa cells as compared with control cells (p < 0.001). Fig. 6(A) displayed the expression of active PARP by immunoblotting. Fig. 6(D) Densitometric analysis revealed an increased activity of PARP in cells treated with IFN-b as compared to control. The cells treated with cisplatin (6 mM) and (12 mM) showed more significant (p < 0.01) increase in cleaved expression of PARP when compared to control, whereas cells treated with combined treatment of low dose cisplatin (6 mM) with IFN-b showed highly significant (p < 0.001) increase in cleaved activity of PARP when compared to control. 3.10. MMP-9 expression was supressed significantly in cells treated with cisplatin and IFN-b Fig. 6(A) showed the blot expression of MMP-9 on cells treatment. Fig. 6(E) exhibited the densitometric analysis, cells treated with cisplatin (12 mM) exhibited more significant inhibition in expression when compared to control, whereas cells treated with IFN-b, cisplatin (6 mM) and combination of cisplatin (6 mM)
The major findings of our study were (i) IFN-b exhibited a synergistic anti-proliferative effect with cisplatin (ii) the combination of these two drugs promoted apoptosis of cervical cancer cells and (iii) IFN-b increased the sensitivity of cisplatin as evident with a reduced dosage of cisplatin. One strategy to overcome chemoresistance and to reduce toxicity is the combination of standard anticancer drugs with naturally occurring compounds with known anticancer activity [20–22]. Cohen et al. and Constantinescu et al. reported the presence of type I IFN binding receptor in HeLa cells [23,24]. The signaling pathway initiated by IFN-b through IFN type I receptors involves STAT1 homodimers and STAT1/STAT2 heterodimers [14]. IFN-b at 1500 IU/mL of drug concentration showed half-maximal inhibition on proliferating HeLa cells, with concomitant increased expression of p-STAT2. The anti-proliferative effect of IFN-b could induce senescence of treated cells via up-regulating tumour suppressor molecules and by activation of STAT. STAT1 and STAT2 were activated by JAK1 and Tyk2 in the cells treated with IFN-b, leading to the formation of transcriptional complexes that translocate to the nucleus to induce expression of certain genes [25]. Ma et al. reported that IFN-b induces suppression of MMP-9 via STAT activation [26], evokes apoptosis via caspase and tumor necrosis factor (TNF-a) related apoptosis inducing ligand (TRAIL) [27]. In the present study, cisplatin exhibited growth inhibition in a dose-dependent manner, with the IC50 value of 12 mM. Inhibition of cell proliferation could be due to intrastrand DNA adducts, activation of mitogen activated protein kinase (MAPK) signal, induction of activated transcription factor3 (ATF3) that activate extracellular signal related kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the p38 kinases [28]. IFN-b synergistically enhanced the growth inhibitory effect of cisplatin on HeLa cells. To our knowledge, we are the first to report that IFN-b (1500 IU/mL) in combination with low doses of cisplatin (0.01–100 mM) resulted in strong synergism in HeLa cells. We observed an increased frequency of micronuclei in cisplatin treated group whereas similar frequency of micronuclei was seen in combination group which has half of the predicted IC50 concentration of cisplatin along with predicted IC50 concentration of IFN-b. The result of our study showed for the first time the combination of cytokine (IFN- b) with cisplatin produces large number of DNA lesions with low dosage of cisplatin. The decreased NDI was observed with cisplatin (12 mM) monotheraphy. Interestingly similar effect was also seen in combination (6 mM of cisplatin and 1500 IU/mL of IFN-b) treatment indicating improved cytostatic effect of cisplatin by IFN-b (Table 2). Several chemotherapeutic agents including paclitaxel, doxorubicin, 5-fluorouracil, cisplatin have been reported to induce NF-kB activation in different cells [29–31]. Kwon et al. showed that inhibition of NF-kB increased chemosensitivity [32]. We demonstrate that low dose of cisplatin in combination of IFN-b inhibited Akt phosphorylation and NF-kB (p65). Synergistic targeting of these pathways has a therapeutic potential for the treatment of primary effusion lymphoma and possibly other malignancies [33]. Genes related to cell survival such as Bcl-2 and Bcl-XL was activated by NF-kB [34]. The down regulated expression levels of anti-apoptotic proteins Bcl-2 and increased expression levels of pro-apoptotic protein Bax in HeLa cells treated with IFN-b and low dosage of cisplatin showed that the drug sensitivity was augmented. It is important to note that IFN-b by itself or in combination with cisplatin disrupted the NF-kB/p-Akt pathway. Activation of STAT has been reported to have a proapoptotic effect
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Fig. 6. Protein expression of Bcl-2, Bax, Cleaved PARP and MMP-9. Cells treated with IFN-b, cisplatin and its combination after 48 h was analysed using western blot. (A) Image of blot. (B, C) Densitometric analysis of the western blot bands for Bcl-2 and Bax was detected, it showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P < 0.001 (***) indicated highly significant. (D) Densitometric analysis of cleaved PARP showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P < 0.001 (***) indicated highly significant. (E) Densitometric analysis of the western blot bands for MMP9 was detected, it showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P< 0.001 (***) indicated highly significant. NS indicated non-significant difference. The results are given as Mean SEM from three experiments. b-actin was used as an internal control.
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through transcription independent interactions with NF-kB (p65) [14]. Chuang et al. systematically reported the baseline levels of NF-kB in carcinoma cell lines when cells were pre-treated with common biologic modulators, NF-kB activation was attenuated significantly and this inhibition may play a role in sensitizing cancer cells to chemotherapeutic drugs [7]. Furthermore, increased PARP cleavage was found in cells treated with combination suggesting increased cell death. NF-kB promotes the survival of cancer cells and also contributes to abnormal proliferation and metastasis [35]. Degradation of extracellular matrix by cancer cells through proteases such as MMPs, cathepsins, and uPA may lead to the separation of the intercellular matrix to promote cancer cell mobility, and may eventually lead to metastasis. MMP-2, MMP-9, and u-PA are most vital for the degradation [36,37]. In our study, we substantiated that low dose of cisplatin with IFN-b suppressed the expression of MMP-9. Yeh et al. and Ji et al. stated that inhibition NF-kB and phosphorylation of ERK1/2 decreases MMP-9 expression, which were similarly observed in our study [38,39]. Thus, as NF-kB is the downstream targets of ERK and AKT pathways, it is suggested from our results that low dose of cisplatin with IFN-b inhibits p-AKT dependent MMP-9 expression probably through resultant suppression of NF-kB activities [40]. 5. Conclusion We concluded that IFN-b suppresses the activation of NF-kB and sensitizes cervical cancer cells to low dose of cisplatin which compromises the drug toxicity and diminishes chemoresistance. These observations may have important pre-clinical implications for the development of new synergistic drug therapeutic regimens based on the combination of cytokine with standard chemotherapeutic agent. Conflict of interests Authors declare no conflict of interest. References [1] R. Siegel, J. Ma, Z. Zou, Cancer statistics, 2014, CA. Cancer J. Clin. 64 (2014) 9–29. [2] F. Muggia, Platinum compounds 30 years after the introduction of cisplatin: implications for the treatment of ovarian cancer, Gynecol. Oncol. 112 (1) (2009) 275–281. [3] J. Reedijk, New clues for platinum antitumor chemistry: kinetically controlled metal binding to DNA, PNAS 100 (7) (2003) 3611–3616. [4] M.P. Barr, S.G. Gray, A.C. Hoffmann, et al., Generation and characterisation of cisplatin resistant non-small cell lung cancer cell lines displaying a stem like signature, PLoS One 8 (2013) e54193. [5] S. Gupta, F. Afaq, H. Mukhtar, Involvement of nuclear factor kappa B, Bax and Bcl-2 in induction of cell cycle arrest and apoptosis by apigenin in human prostate carcinoma cells, Oncogene 21 (23) (2002) 3727–3738. [6] B.B. Aggarwal, Nuclear factor-kappaB: the enemy within, Cancer Cell 6 (3) (2004) 203–208. [7] S.E. Chuang, P.Y. Yeh, Y.S. Lu, et al., Basal levels and patterns of anticancer druginduced activation of nuclear factor-kappaB (NF-kappaB), and its attenuation by tamoxifen, dexamethasone, and curcumin in carcinoma cells, Biochem. Pharmacol. 63 (2002) 1709–1716. [8] J. Li, G. Lau, L. Chen, et al., Interleukin 23 promotes hepatocellular carcinoma metastasis via NF-kappa B induced matrix metalloproteinase 9 expression, PLoS One 7 (2012) e46264. [9] R.H. Chou, S.C. Hsieh, Y.L. Yu, et al., Fisetin inhibits migration and invasion of human cervical cancer cells by downregulating urokinase plasminogen activator expression through suppressing the p38 MAPK-dependent NFkappaB signaling pathway, PLoS One 8 (2013) e71983. [10] C.Y. Hsieh, P.C. Tsai, C.L. Chu, et al., Brazilein suppresses migration and invasion of MDA-MB-231 breast cancer cells, Chem. Biol. Interact. 204 (2013) 105–115. [11] F. Li, G. Sethi, Targeting transcription factor NF-kB to overcome chemoresistance and radioresistance in cancer therapy, Biochim. Biophys. Acta 1805 (2010) 167–180.
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Interferon b improves the efficacy of low dose cisplatin by inhibiting NF-kB/p-Akt signaling on HeLa cells Purushoth Ethiraja , Karpagam Veerappanb , Shila Samuelb , Sundaresan Sivapathama,* a b
Department of Medical Research, SRM Medical College Hospital and Research Centre, SRM University, Kattankulathur 603203, Tamilnadu, India Department of Biochemistry, VRR Institute of Biomedical Science (Affiliated to University of Madras), Chennai 600 056, India
A R T I C L E I N F O
Article history: Received 19 January 2016 Received in revised form 26 April 2016 Accepted 26 April 2016 Keywords: Cisplatin Interferon b Chemosensitivity NF-kB Akt PARP
A B S T R A C T
The purpose of this study was to evaluate the anticancer efficacy of interferon b in combination with low dose of cisplatin on human cervical cancer progression, as well as its principal action mechanism. The combination treatment synergistically potentiated the effect of interferon b on cell growth inhibition and DNA damage on HeLa cells by repressing NF-kB/p-Akt signaling. Synergistic targeting of these pathways has a therapeutic potential. Further, the combination treatment ameliorated the expression of proapoptotic Bax, and decreased the expression of anti-apoptotic protein Bcl-2. Additionally, the expression of active PARP was significantly increased and MMP-9 level was decreased in combination group as compared to the expression seen for the treatment with interferon b or cisplatin alone. Results demonstrate that the synergistic inhibitory effects of interferon b and low dose of cisplatin on human cervical cancer cells and also suggest that the inhibition of NF-kB/p-Akt signaling pathway plays a critical role in the anticancer effects of combination treatment along with the induction of PARP. Therefore, the combination of interferon b and cisplatin may be a useful treatment for human cervical cancer, with a greater effectiveness than other treatments. ã 2016 Elsevier Masson SAS. All rights reserved.
1. Introduction Cervical cancer is the second most frequent cause of cancer in women [1]. Cisplatin is an effective agent in its treatment [2]. However, its usage is limited by its toxicity and acquired chemoresistance through the course of treatment [3]. Barr et al. stated that increased doses of cisplatin treatment for long term on lung cancer cell lines, led to cell lines resistant to cisplatin mediated cell death [4]. Activation of p-Akt and the NF-kB/Bcl-2 pathways leads to inhibition of chemotherapy-induced apoptosis, which results in treatment resistance [5,6]. Activation of NF-kB has been identified as a key mechanism of cisplatin resistance. NF-kB activity is inversely correlated with cellular sensitivity to chemotherapy in carcinoma cell lines [7]. NF-kB promotes the migration and metastasis of hepatocellular carcinoma cells [8], cervical cancer cells [9], and breast cancer cells [10] through an upregulation of Matrix Metalloproteinases (MMPs). Suppression of NF-kB activation is effective in the prevention and treatment of cancer [6]. Li and Sethi reported that chemoresistance is mediated
* Corresponding author. E-mail address: [email protected] (S. Sivapatham). http://dx.doi.org/10.1016/j.biopha.2016.04.058 0753-3322/ ã 2016 Elsevier Masson SAS. All rights reserved.
through several genes regulated by NF-kB and inhibition of this transcription factor increases sensitivity of cancer cells to the apoptotic action of chemotherapeutic agents [11]. Thus, it was hypothesised that agents inhibiting the activation of NF-kB may exhibit the therapeutic potential for the suppression of carcinogenesis and tumor metastasis [12]. The antitumor effects of interferon b (IFN-b) have been attributed to the inhibition of cell proliferation, induction of cell differentiation, immunomodulation and alteration of the level of gene expression in target cells [13]. The binding of IFN-b to the type I IFN receptor results in the phosphorylation and activation of tyrosine kinase2 (Tyk2) and Janus kinase1 (JAK1), which in turn regulates the phosphorylation and activation of signal transducers and activators of transcription (STAT) [14]. Josiane et al. reported that IFN-induced IRF1 (interferon regulatory factor-1) inhibited NF-kB dependent activation of MMP-9 by binding to the promoter region overlapping the NF-kB binding site [15]. Li et al. demonstrated that IFN-b produced synergistic anti-tumour effects with cisplatin through up-regulated p53 expression on mesothelioma cells [16]. Hence to improve the sensitivity and efficacy of cisplatin at lower doses it is combined with IFN-b to achieve synergistic effect. We previously reported the synergistic anti-carcinogenic effect of IFN-b with cisplatin on human breast adenocarcinoma
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MDA MB231 cells [17]. In this study, we aimed to evaluate the effect of IFN-b individually and in combination with low of dose cisplatin on cellular proliferation, genotoxicity, and apoptosis in HeLa cells. 2. Materials and methods 2.1. Drugs and reagents Interferon b Human (Cat No.I4151, Purity 95%), platinum(II) (Cat No.479306), cytochalasin B (Cat No. C6762), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Cat No. M2003) and dimethyl sulphoxide (DMSO) (Cat No.D2438) were purchased from Sigma Aldrich (Sigma Aldrich Co., USA). Antibodies against STAT2, p-STAT2 (Tyr690), Bax, Bcl-2, p-Akt (Ser473), NF-kB (p65), PARP and MMP9 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against IkB and p-IkB were purchased from Cell Signaling Technology, Inc (MA, USA). Horseradish peroxidase conjugated anti-mouse was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2.2. Maintenance of cell lines The cell lines HeLa obtained from National Centre for Cell Science (Pune, India) were maintained and grown in a humidified incubator at 37 C with 5% CO2. Cells were grown as a monolayer in plastic tissue culture flasks in Dulbecco’s Modified Eagles Medium (DMEM) (GIBCO, Grand Island, New York, USA). The media was supplemented with 10% fetal bovine serum (FBS) (GIBCO, Grand Island, New York, USA) and antibiotics (Penicillin 50 IU/mL, Streptomycin 3.5 mg/mL and Gentamycin 2.5 mg/mL) (GIBCO, Grand Island, New York, USA).
2.3. Cell proliferation assay HeLa cells were seeded in 96-well plates at a density of 5 103 cells/well in 200 mL DMEM containing 10% FBS and incubated overnight. Nonadherent cells were removed by gentle washing after 24 h. Then cells were replaced with serum free medium with varying concentrations of interferon b (100–2500 IU/mL) and cisplatin (0.01–100 mM). A negative control containing serum free medium with DMSO was also evaluated. After 72 h of treatment, the plates were incubated with 20 mL 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) solutions (5 mg/mL) for 3 h at 37 C. The formazan was dissolved in 150 mL/well dimethyl sulfoxide (DMSO) and the absorbance was detected at 590 nm using microplate reader (Bio Rad, USA). Cell viability was expressed as a percentage of untreated cells, which served as the negative control group and was designated as 100%; the results were expressed as a percentage of the negative control. The median inhibitory concentration (IC50) (defined as the drug concentration at which cell growth was inhibited by 50%) was assessed from the dose response curves.
2.4. Synergistic analysis The synergistic effect of IFN-b with cisplatin was examined using median effect method as originally described by Chou and Talalay [18]. The combination index (CI) values reflect the nature of the interaction between IFN-b and cisplatin. The CI < 1 indicates synergy, CI = 1, addictive, CI > 1, antagonism. The combination index analysis was performed using Compusyn software (comboSyn, Inc., Paramus, (NJ)).
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2.5. Tryphan blue dye exclusion assay HeLa cells were treated with IFN-b and cisplatin at 1500 IU/mL and 12 mM, respectively. The low dosage of cisplatin (6 mM) was also studied. For the combination, IFN-b (1500 IU/mL) and cisplatin (6 mM half of the IC50) were treated for 72 h. Cell viability was determined by the tryphan blue dye exclusion assay; this dye excluded living cells and only penetrated the cell membrane of dead cells. The proportion of dead cells was determined by counting the number of stained cells using a hemocytometer. 2.6. Western blotting The cells were seeded in 24-well plates at a density of 5 104 cells/well in 1 mL of DMEM containing 10% FBS overnight. Nonadherent cells were removed by gentle washing after 24 h. Then, cells were treated with interferon b (1000 & 1500 IU/mL) for STAT and p-STAT2 (Tyr690). The cells were treated with interferon b and cisplatin at 1500 IU/mL and 12 mM respectively, for combination, 1500 IU/mL of IFN-b and half of the obtained concentration of IC50 (6 mM) of cisplatin were used for t-Akt, p-Akt (Ser473), IkB, pIkB, NF-kB (p65), Bax, Bcl-2, PARP, MMP-9. A negative control containing serum free medium was also evaluated. After 48 h of treatment, cells were lysed by the addition of cold RIPA buffer [150 mM NaCl, 50 mM Tris HCL, 0.1% SDS, 1% Triton X-100, 1 mM PMSF, 2 mM NaF, Na3VO4, b-glycerophosphate and 2 mM EDTA, and fresh protease inhibitor cocktail (Cat No. P8340, Sigma Aldrich)] and cell lysate was centrifuged at 14,000 rpm at 4 C for 20 min. The supernatant was harvested and analysed for protein content using BCA method (Cat No. 23227, Pierce, USA). Protein was denatured in sample buffer, then separated on 12% SDS-PAGE, and transferred to polyvinylidene difluoride membranes (semidry trans-blot system). The blots were blocked for 2 h at room temperature with Tris-Buffered Saline (TBS, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl) containing 5% non-fat milk. The blots were washed three times with TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.02% Tween 20) and incubated with specific primary antibodies (1:1000 dilutions) at 4 C overnight. The blots were incubated for 1 h at room temperature with secondary antibodies (1:5000 dilutions), and detected by ECL detection reagent. To ensure that equal amounts of sample protein were applied for electrophoresis, b-actin was used as an internal control. Densitometric analysis was done using ImageJ software. 2.7. Analysis of DNA damage using micronucleus assay The cytotoxicity test (MTT) demonstrated that IFN-b and cisplatin at 1500 IU/mL and 12 mM respectively produced 50% reduction in cell viability (IC50). These concentrations were used for micronuclei (MN) analysis. For combination, 1500 IU/mL of IFNb and half of the obtained IC50 concentration (6 mM) of cisplatin were used. The MN assay was performed using a slight modification of previously published method of Fenech [19]. Cells were seeded at 1 105 in 60 mm dishes. After 24 h, nonadherant cells were removed by gentle washing with PBS and then cells were treated with concentrations as mentioned above, alone and in combination. Cytochalasin-B (3 mg/mL) was added at 44th h to arrest cells at the cytokinesis stage. The cells were harvested after 72 h. The cells were subjected to a mild treatment with hypotonic solution (0.45% KCl). Then, the cells were centrifuged (1000 rpm, 10 min) and fixed three times in cold methanol and acetic acid (3:1). The initial addition of fixative contained 1% formaldehyde, which enhanced cytoplasm preservation. The cells were dropped on cold glass slides, stained with 10% Giemsa solution (pH 6.8, GIBCO, Burlington, USA) for 5 min. The slides were coded and scored
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under a light microscopy at 400* magnification or 1000* as required. Thousand binucleated cells (cells with two daughter nuclei surrounded by cytoplasm) were scored for the presence of MN according to the scoring criteria given by Fenech [19]. Based on the recorded data, the frequency of micronuclei (MNi) was calculated using the formula MNi = a/b and the error was calculated by p MNi = a/b where ‘a’ is the total number of micronuclei and ‘b’ is the total number (1000) of binucleated cells scored. The ability of the cells to proliferate in vitro was evaluated by counting the number of cells with one, two and four MN on the same slide using the nuclear division index (NDI), calculated
according to the formula: NDI = (MI + 2MII + 3MIII + 4MIV)/N, where MI to MIV represent the number of cells with one to four nuclei, respectively, and N is the number of cells scored [19].
2.8. Statistical analysis Data are presented as Mean SEM. Each value is the mean of at least three separate experiments. Statistical evaluation was performed using an unpaired Student’s t-test. p values of <0.05 was considered to be statistically significant.
Fig. 1. HeLa cells were treated with cisplatin and IFN-b cell proliferation was assessed using the MTT assay. Cells were treated with various concentrations of Cisplatin (0.01– 100 mM) (A) Interferon b (100–2500 IU/ml) (B) and combination of cisplatin (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50 100) with IFN-b (1500 IU/ml) (C). Cell proliferation was determined by MTT. Data represented are Mean SEM (n = 3).
P. Ethiraj et al. / Biomedicine & Pharmacotherapy 82 (2016) 124–132 Table 1 Synergistic combinatory effect of cisplatin with IFN-b. Cells were treated with varying concentrations of cisplatin (0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100) + IFN-b (1500 IU/ml). CI values between 0.1–0.3 shows strong synergism. Cisplatin (mM)
IFN-b (IU/ml)
CI Values
0.01 0.05 0.1 0.5 1 5 10 50 100
1500 1500 1500 1500 1500 1500 1500 1500 1500
0.784 0.721 0.681 0.592 0.351 0.331 0.281 0.246 0.212
CI indicates combination index.
3. Results 3.1. Inhibition of growth in HeLa cells by IFN-b and its synergistic effect with anticancer agent cisplatin The effects of IFN-b and cisplatin on the proliferation of HeLa cells were examined. The percentage of viable cells obtained in the MTT assay with varying concentrations of
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cisplatin (0.01–100 mM) and IFN-b (100–2500 IU/mL) were shown in Fig. 1(A and B). Dramatic dose-dependent reduction on the cell proliferation was observed in both IFN-b and cisplatin treated cells after 72 h of exposure. The IC50 values of interferon b and cisplatin were 1500 IU/mL and 12 mM respectively. Synergistic effect (CI < 1) on anti-proliferation was observed when different concentrations of cisplatin was combined with 1500 IU/mL of IFN-b in Table 1. 3.2. IFN-b induced p-STAT2 (Try690) signaling required for inhibition of cell proliferation Fig. 2(A) showed the protein expression IFN-b induced STAT2 and p-STAT2 (Try690) signaling mediated cell proliferation inhibition on cells treated with 1000 and 1500 IU/mL. Densitometric analysis showed a significant difference in the expression of p-STAT2 in cells treated with 1500 IU/mL of IFN-b compared to control (Fig. 2(B)). 3.3. Effects of cisplatin/IFN-b and its synergistic combination on HeLa cell death To investigate the effects of IFN-b and/or cisplatin on survival or growth, HeLa cells were exposed with 1500 IU/mL of IFN-b,
Fig. 2. Protein expression of STAT2 and p-STAT2 (Try690) on cells treated with IFN-b. Hela cells were treated with 1000 and 1500 IU/mL (Dose of IC50) of IFN-b, whereas untreated cells were used as control (0), after 48 h protein expression of STAT2 and p-STAT2 was analysed using western blot. (A) Representative image of blot. (B) Densitometric analysis of p-STAT2 was normalized with STAT2 and represented as ratio (p-STAT2/STAT2), it showed significant difference compared to control. P < 0.05 (*) indicated significant difference, whereas p < 0.01 (**) showed more significance. The results are given as Mean SEM from three experiments. b-actin was used as an internal control.
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Fig. 3. Cell death caused by cisplatin and/or IFN-b treatment on HeLa cells. Percentage of cell death was significantly increased P < 0.05 (*) in cells treated with IFN-b and cisplatin (6 mM), whereas its shows more significant difference P < 0.01 (**) at cisplatin (12 mM), and highly significant p < 0.001 (***) increase at combination (Cisplatin (6 mM) + IFN-b (1500 IU/mL)) group when compared to control.
Fig. 4. Frequency of Micronuclei. The binucleated cells with micronucleus found after treatment of the predicted dose of IFN-b, cisplatin and its combination (Cisplatin (6 mM) + IFN-b (1500 IU/mL)) showed a significant difference, when compared to control. P < 0.05 (*) indicates significant difference, p < 0.01 (**) indicates more significant, whereas p < 0.001 (***) indicates highly significant difference. NS indicates Non-significant difference.
cisplatin at 6 and 12 mM and its combination for 72 h. Fig. 3 showed that the percentage of cell death was significantly increased (P < 0.05) in cells treated with IFN-b at 1500 IU/mL and cisplatin at 6 mM respectively, whereas more significant difference (P < 0.01) was observed in cells treated with cisplatin (12 mM) when compared to control. Highly significant (p < 0.001) cell death was observed at combination group, when compared to control.
3.5. NDI Table 2 represents NDI of control and treated cells. The cells treated with IFN-b and cisplatin alone showed a significant difference (p < 0.05) when compared to control. The NDI value of the combination group was 1.032 0.07 more significant (p < 0.01), compared to control.
3.4. Induction of micronuclei frequency on cells treated with IFN-b and cisplatin alone and in combination Fig. 4 showed the graphical representation of the micronuclei frequencies. Baseline micronuclei frequency obtained in untreated control cells was 0.018 0.004. There was no significant difference between vehicle control groups (DMSO) and control groups. IFN-b treated cells induced significant increase in MN frequencies when compared to control. Lower dosage of cisplatin (6 mM) induced MN frequencies were more significant (p < 0.01) compared to control. The micronuclei frequencies obtained in the cisplatin (12 mM) treated group was (0.0919 0.98) highly significant (p < 0.001), compared to control. The combinational group also showed highly significant difference among MN frequencies, when compared to control. There was no significant change when cisplatin group (12 mM) compared with combination group (6 mM cisplatin + 1500 IU/mL IFN-b).
Table 2 Illustrates obtained nuclear division index (NDI) values. Mean and standard deviation of nuclear division index (NDI) for 1500 cells analysed after treatment of HeLa cells to the predicted dose of IFN-b and cisplatin showed a significant difference P < 0.05 (*) when compared to control, whereas the combination (Cisplatin (6 mM) + IFNb (1500 IU/mL)) group showed more significant p < 0.01 (**). Control DMSO IFN-b Cisplatin (6 mM) Cisplatin (12 mM) Combination * **
p < 0.05. p < 0.01.
1.5122 0.11 1.516 0.12 1.272 0.08* 1.202 0.09* 1.120 0.10* 1.032 0.07**
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Fig. 5. Protein expression of Akt, p-Akt (Ser473), IkB, p- IkB, NF-kB on cells treated with cisplatin, IFN-b and its combination after 48 h was analysed using western blot. (A) Image of blot. (B) Densitometric analysis of p-Akt was normalized to t-Akt and represented as ratio (p-Akt/Akt), it showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P < 0.001 (***) indicated highly significant. (C) Densitometric analysis of p-IkB was normalized to t-IkB and represented as ratio (p-IkB/IkB), it showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P < 0.001 (***) indicated highly significant. (D) Densitometric analysis of NF-kB showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P < 0.001 (***) indicated highly significant. NS indicated non-significant difference. The results are given as Mean SEM from three experiments. b-actin was used as an internal control.
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3.6. IFN-b prevents cisplatin induced up-regulation of p-Akt(Ser473) in cells
and IFN-b displayed highly significant suppression on the expression of MMP-9 compared to control.
The protein expression of Akt and p-Akt (Ser473) on treated cells were studied. Fig. 5(A) exhibited the blot expression of Akt and p-Akt. Fig. 5(B) densitometric analysis exhibited cells treated with cisplatin (12 mM) showed non-significant change when compared to control. Cells treated with IFN-b and cisplatin (6 mM) showed more significant (p < 0.01) and significant (p < 0.05) down regulation in p-Akt respectively, when compared to control. The expression of p-Akt exhibited highly significant (p < 0.001) at combination group when compared to control.
4. Discussion
3.7. The diminished expression of p-IkB at combined treatment was observed in cells The protein expression of IkB and p-IkB on treated cells was studied. Fig. 5(A) exhibited the protein expression of IkB and p-IkB. Fig. 5(C) Densitometric analysis exhibited cells treated with cisplatin (12 mM) showed non-significant change when compared to control. Cells treated with IFN-b and cisplatin (6 mM) showed more significant (p < 0.01) down regulation in p-IkB when compared to control. The reduced expression level of p-IkB was significantly (p < 0.001) high at combination group when compared to control. 3.8. Inhibition of NF-kB (p65) expression was observed in cells treated with low dose of cisplatin with IFN-b The protein expression of NF-kB (p65) on treated cells was studied. Fig. 5(A) exhibited the protein expression of p65. Fig. 5(D) Densitometric analysis revealed cells treated with cisplatin (12 mM) showed non-significant change when compared to control. Cells treated with IFN-b and cisplatin (6 mM) showed significant (p < 0.05) and more significant (p < 0.01) down regulation in p65 when compared to control, respectively. The expression level of p65 was highly significant (p < 0.001) at reduced dosage of cisplatin (6 mM) when compared to control. 3.9. IFN-b and low dose of cisplatin sensitizes cervical cancer cells through Bcl-2 family members and induced the activity of PARP Fig. 6(A) showed the expression of Bcl-2 and Bax by immunoblotting. Fig. 6(B and C) densitometric analysis showed that IFN-b and low dose of cisplatin (6 mM) is able to markedly down-regulate the expression of Bcl-2 and increase the expression Bax in HeLa cells as compared with control cells (p < 0.001). Fig. 6(A) displayed the expression of active PARP by immunoblotting. Fig. 6(D) Densitometric analysis revealed an increased activity of PARP in cells treated with IFN-b as compared to control. The cells treated with cisplatin (6 mM) and (12 mM) showed more significant (p < 0.01) increase in cleaved expression of PARP when compared to control, whereas cells treated with combined treatment of low dose cisplatin (6 mM) with IFN-b showed highly significant (p < 0.001) increase in cleaved activity of PARP when compared to control. 3.10. MMP-9 expression was supressed significantly in cells treated with cisplatin and IFN-b Fig. 6(A) showed the blot expression of MMP-9 on cells treatment. Fig. 6(E) exhibited the densitometric analysis, cells treated with cisplatin (12 mM) exhibited more significant inhibition in expression when compared to control, whereas cells treated with IFN-b, cisplatin (6 mM) and combination of cisplatin (6 mM)
The major findings of our study were (i) IFN-b exhibited a synergistic anti-proliferative effect with cisplatin (ii) the combination of these two drugs promoted apoptosis of cervical cancer cells and (iii) IFN-b increased the sensitivity of cisplatin as evident with a reduced dosage of cisplatin. One strategy to overcome chemoresistance and to reduce toxicity is the combination of standard anticancer drugs with naturally occurring compounds with known anticancer activity [20–22]. Cohen et al. and Constantinescu et al. reported the presence of type I IFN binding receptor in HeLa cells [23,24]. The signaling pathway initiated by IFN-b through IFN type I receptors involves STAT1 homodimers and STAT1/STAT2 heterodimers [14]. IFN-b at 1500 IU/mL of drug concentration showed half-maximal inhibition on proliferating HeLa cells, with concomitant increased expression of p-STAT2. The anti-proliferative effect of IFN-b could induce senescence of treated cells via up-regulating tumour suppressor molecules and by activation of STAT. STAT1 and STAT2 were activated by JAK1 and Tyk2 in the cells treated with IFN-b, leading to the formation of transcriptional complexes that translocate to the nucleus to induce expression of certain genes [25]. Ma et al. reported that IFN-b induces suppression of MMP-9 via STAT activation [26], evokes apoptosis via caspase and tumor necrosis factor (TNF-a) related apoptosis inducing ligand (TRAIL) [27]. In the present study, cisplatin exhibited growth inhibition in a dose-dependent manner, with the IC50 value of 12 mM. Inhibition of cell proliferation could be due to intrastrand DNA adducts, activation of mitogen activated protein kinase (MAPK) signal, induction of activated transcription factor3 (ATF3) that activate extracellular signal related kinases (ERKs), c-Jun N-terminal kinases (JNKs) and the p38 kinases [28]. IFN-b synergistically enhanced the growth inhibitory effect of cisplatin on HeLa cells. To our knowledge, we are the first to report that IFN-b (1500 IU/mL) in combination with low doses of cisplatin (0.01–100 mM) resulted in strong synergism in HeLa cells. We observed an increased frequency of micronuclei in cisplatin treated group whereas similar frequency of micronuclei was seen in combination group which has half of the predicted IC50 concentration of cisplatin along with predicted IC50 concentration of IFN-b. The result of our study showed for the first time the combination of cytokine (IFN- b) with cisplatin produces large number of DNA lesions with low dosage of cisplatin. The decreased NDI was observed with cisplatin (12 mM) monotheraphy. Interestingly similar effect was also seen in combination (6 mM of cisplatin and 1500 IU/mL of IFN-b) treatment indicating improved cytostatic effect of cisplatin by IFN-b (Table 2). Several chemotherapeutic agents including paclitaxel, doxorubicin, 5-fluorouracil, cisplatin have been reported to induce NF-kB activation in different cells [29–31]. Kwon et al. showed that inhibition of NF-kB increased chemosensitivity [32]. We demonstrate that low dose of cisplatin in combination of IFN-b inhibited Akt phosphorylation and NF-kB (p65). Synergistic targeting of these pathways has a therapeutic potential for the treatment of primary effusion lymphoma and possibly other malignancies [33]. Genes related to cell survival such as Bcl-2 and Bcl-XL was activated by NF-kB [34]. The down regulated expression levels of anti-apoptotic proteins Bcl-2 and increased expression levels of pro-apoptotic protein Bax in HeLa cells treated with IFN-b and low dosage of cisplatin showed that the drug sensitivity was augmented. It is important to note that IFN-b by itself or in combination with cisplatin disrupted the NF-kB/p-Akt pathway. Activation of STAT has been reported to have a proapoptotic effect
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Fig. 6. Protein expression of Bcl-2, Bax, Cleaved PARP and MMP-9. Cells treated with IFN-b, cisplatin and its combination after 48 h was analysed using western blot. (A) Image of blot. (B, C) Densitometric analysis of the western blot bands for Bcl-2 and Bax was detected, it showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P < 0.001 (***) indicated highly significant. (D) Densitometric analysis of cleaved PARP showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P < 0.001 (***) indicated highly significant. (E) Densitometric analysis of the western blot bands for MMP9 was detected, it showed significant difference compared to control. P < 0.05 (*) indicated significant difference, p < 0.01 (**) showed more significant, whereas P< 0.001 (***) indicated highly significant. NS indicated non-significant difference. The results are given as Mean SEM from three experiments. b-actin was used as an internal control.
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through transcription independent interactions with NF-kB (p65) [14]. Chuang et al. systematically reported the baseline levels of NF-kB in carcinoma cell lines when cells were pre-treated with common biologic modulators, NF-kB activation was attenuated significantly and this inhibition may play a role in sensitizing cancer cells to chemotherapeutic drugs [7]. Furthermore, increased PARP cleavage was found in cells treated with combination suggesting increased cell death. NF-kB promotes the survival of cancer cells and also contributes to abnormal proliferation and metastasis [35]. Degradation of extracellular matrix by cancer cells through proteases such as MMPs, cathepsins, and uPA may lead to the separation of the intercellular matrix to promote cancer cell mobility, and may eventually lead to metastasis. MMP-2, MMP-9, and u-PA are most vital for the degradation [36,37]. In our study, we substantiated that low dose of cisplatin with IFN-b suppressed the expression of MMP-9. Yeh et al. and Ji et al. stated that inhibition NF-kB and phosphorylation of ERK1/2 decreases MMP-9 expression, which were similarly observed in our study [38,39]. Thus, as NF-kB is the downstream targets of ERK and AKT pathways, it is suggested from our results that low dose of cisplatin with IFN-b inhibits p-AKT dependent MMP-9 expression probably through resultant suppression of NF-kB activities [40]. 5. Conclusion We concluded that IFN-b suppresses the activation of NF-kB and sensitizes cervical cancer cells to low dose of cisplatin which compromises the drug toxicity and diminishes chemoresistance. These observations may have important pre-clinical implications for the development of new synergistic drug therapeutic regimens based on the combination of cytokine with standard chemotherapeutic agent. Conflict of interests Authors declare no conflict of interest. References [1] R. Siegel, J. Ma, Z. Zou, Cancer statistics, 2014, CA. Cancer J. Clin. 64 (2014) 9–29. [2] F. Muggia, Platinum compounds 30 years after the introduction of cisplatin: implications for the treatment of ovarian cancer, Gynecol. Oncol. 112 (1) (2009) 275–281. [3] J. Reedijk, New clues for platinum antitumor chemistry: kinetically controlled metal binding to DNA, PNAS 100 (7) (2003) 3611–3616. [4] M.P. Barr, S.G. Gray, A.C. Hoffmann, et al., Generation and characterisation of cisplatin resistant non-small cell lung cancer cell lines displaying a stem like signature, PLoS One 8 (2013) e54193. [5] S. Gupta, F. Afaq, H. Mukhtar, Involvement of nuclear factor kappa B, Bax and Bcl-2 in induction of cell cycle arrest and apoptosis by apigenin in human prostate carcinoma cells, Oncogene 21 (23) (2002) 3727–3738. [6] B.B. Aggarwal, Nuclear factor-kappaB: the enemy within, Cancer Cell 6 (3) (2004) 203–208. [7] S.E. Chuang, P.Y. Yeh, Y.S. Lu, et al., Basal levels and patterns of anticancer druginduced activation of nuclear factor-kappaB (NF-kappaB), and its attenuation by tamoxifen, dexamethasone, and curcumin in carcinoma cells, Biochem. Pharmacol. 63 (2002) 1709–1716. [8] J. Li, G. Lau, L. Chen, et al., Interleukin 23 promotes hepatocellular carcinoma metastasis via NF-kappa B induced matrix metalloproteinase 9 expression, PLoS One 7 (2012) e46264. [9] R.H. Chou, S.C. Hsieh, Y.L. Yu, et al., Fisetin inhibits migration and invasion of human cervical cancer cells by downregulating urokinase plasminogen activator expression through suppressing the p38 MAPK-dependent NFkappaB signaling pathway, PLoS One 8 (2013) e71983. [10] C.Y. Hsieh, P.C. Tsai, C.L. Chu, et al., Brazilein suppresses migration and invasion of MDA-MB-231 breast cancer cells, Chem. Biol. Interact. 204 (2013) 105–115. [11] F. Li, G. Sethi, Targeting transcription factor NF-kB to overcome chemoresistance and radioresistance in cancer therapy, Biochim. Biophys. Acta 1805 (2010) 167–180.
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