Molecular and Cellular Biochemistry https://doi.org/10.1007/s11010-018-3278-z

Decitabine augments cytotoxicity of cisplatin and doxorubicin to bladder cancer cells by activating hippo pathway through RASSF1A Madhuram Khandelwal1 · Vivek Anand1 · Sandeep Appunni1 · Amlesh Seth2 · Prabhjot Singh2 · Sandeep Mathur3 · Alpana Sharma1 Received: 12 July 2017 / Accepted: 16 January 2018 © Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract Genetic abnormalities and epigenetic alterations both play vital role in initiation as well as progression of cancer. Whereas genetic mutations cannot be reversed, epigenetic alterations such as DNA methylation can be reversed by the application of DNA methyltransferase inhibitor decitabine. Epigenetic silencing of RASSF1A and involvement of hippo pathway both have been shown to involve in chemo-resistance. Purpose of this study was to observe the effect of combination treatment of decitabine with cisplatin or doxorubicin on bladder cancer cells involving hippo pathway through RASSF1A. Bladder cancer cells (HT1376 & T24) were treated with decitabine and its effect on RASSF1A expression, hippo pathway molecules (MST & YAP), and its downstream targets (CTGF, CYR61 & CTGF) was observed. Effect of decitabine pretreatment on sensitivity of bladder cancer cells towards chemotherapeutic drugs was also studied. Decitabine treatment leads to restoration of RASSF1A, activation of hippo pathway followed by decreased expression of its oncogenic downstream targets (CTGF & CYR61). Further pretreatment of decitabine enhanced cytotoxicity of cisplatin and doxorubicin to bladder cancer cells. Keywords  Urinary bladder cancer · RASSF1A · Hippo pathway · CTGF, CYR61, Chemosensitivity · Cytotoxicity

Introduction Urothelial bladder carcinoma (UBC) is a common malignancy of urinary tract currently ranked as 9th most common cancer [1]. According to GLOBOCAN (2012), 430,000 new bladder cancer cases and 165,000 bladder cancer deaths occurred worldwide, with predominance of 75% of total in men [2]. Most of bladder cancer patients present with superficial cancer and have a favorable prognosis, out of them, 10–20% further progress into invasive cancer and requires systemic chemotherapy as treatment option [3]. Use of cisplatin-based chemotherapy as first-line treatment regimen such as (M-VAC) methotrexate, vinblastine, doxorubicin and cisplatin, dose-dense M-VAC, and gemcitabine and cisplatin (GC) has shown to use with efficacy in treating metastatic transitional cell carcinoma (TCC) of urothelium * Alpana Sharma [email protected] 1



Department of Biochemistry, AIIMS, New Delhi, India

2



Department of Urology, AIIMS, New Delhi, India

3

Department of Pathology, Aiims, New Delhi, India



[4, 5]. Apart from combination of M-VAC, doxorubicin is also employed as an agent of intravesical instillation into the bladder or by intravenous injection. Many mechanisms of cisplatin and doxorubicin resistance have been proposed including changes in uptake and efflux of drug, detoxifying enzymes, and apoptosis-related genes [6, 7]. These treatment regimens have shown initial high response rate of 40–70% with 8 months of median progression-free survival and 15 months of overall survival, indicating a highly chemo-resistant disease upon relapse [8]. Although bladder cancer is a chemotherapy-sensitive malignancy, even after treating with chemotherapy majority of patients develop disease progression. Currently, there is no standard of care for patients in second-line setting, i.e., after progression on cisplatin-based chemotherapy. Genetic and epigenetic alterations both play a critical role in tumorigenesis. Epigenetic silencing of genes through DNA methylation influences tumorigenesis, response to drug therapy and is one of the main causes of drug resistance, thus can be used as an attractive target for epigenetic drug therapy [9]. RASSF1A is a tumor suppressor gene, its decreased expression due to the promoter hypermethylation is one of the most frequent epigenetic inactivation event

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observed in many tumors including bladder cancer [10]. RASSF1A through its SARAH domain interact with MST leads to activation of hippo pathway followed by decreased expression of downstream oncogenic targets and act as an upstream regulator of this tumor suppressor pathway [11, 12]. Studies have reported involvement of RASSF1A [13, 14] and hippo pathway molecules MST, YAP, CTGF, and CYR61 in chemo-resistance [15–17]. Demethylating agent decitabine has been shown to restore expression of silenced genes by reversing methylation and suppressed growth of tumors in vitro. Although therapeutic efficacy of decitabine has been established in hematological cancers, few studies have shown its usefulness in solid tumors [18–20]. Therefore, our aim was to assess utility of decitabine along with chemotherapeutic agent as an improved treatment option for UBC. We hypothesized that treatment with decitabine by restoring expression of RASSF1A or by decreasing expression of oncogenic downstream targets of hippo pathway may augment cytotoxic effects of chemotherapeutic drugs, thus help in reducing chemo-resistance.

Materials and methods Cell culture and reagents Two bladder cancer cell lines HT1376 and T24 were used in the study. HT1376 obtained from American Type Culture Collection (ATCC, US), T24 from NCCS Pune, and HeLa was kind gift from Dr. P. P. Chattopadhyay (Department of Biochemistry, AIIMS). Cells were cultured in complete medium and maintained in a humidified atmosphere containing 5% ­CO2 at 37 °C. Decitabine (5-Aza-CdR), Cisplatin, and Doxorubicin were purchased from Sigma-Aldrich (U.S.). Antibodies used for western blots, like RASSF1A and pMST were from Abcam (UK), MST, YAP, pYAP, and AREG from ThermoFischer (US), CTGF, CYR61, and beta-actin were from Santa Cruz. Primers were designed using Primer3 software and ordered from Sigma-Aldrich (US). All in vitro assays were done in triplicates.

Methylation‑specific PCR (MSPCR) of RASSF1A Genomic DNA was isolated from cultured cells using DNA extraction Kit (Qiagen, Germany). DNA was modified with bisulfite reagent using the Epitech DNA Modification Kit (Qiagen, Germany) and amplified using primers specific for the methylated and unmethylated sequence of RASSF1A. Methylated Forward: 5′GGG TTT TGC GAG AGC GCG3′ Reverse: 5′GCT AAC AAC GCG AAC CG3′; Unmethylated Forward 5′GGT​TTT​GTG​AGA​GTG​TGT​TTAG3′ Reverse:

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5′CACT ​ AAC ​ AAA ​ CAC ​ AAA ​ CCA ​ AAC3′. The amplification products were analyzed by 2% agarose gel electrophoresis.

Q‑PCR of RASSF1A Total RNA from cells were extracted with TRIzol reagent (Sigma, USA). 1  µg of RNA was used for synthesis of cDNA, which later used as a template to analyze the gene expression (using BIO-RAD CFX96 Touch™ Real-Time PCR Detection System) with specific primers using Maxima SYBR green master mix (Fermentas, USA). Primers used for RASSF1A were Forward: 5′CTC GTC TGC CTG GAC TGT TG3′, Reverse: 5′GAT GAA GCC TGT GTA AGA ACC G3′. ∆Ct values were calculated by target gene Ct minus housekeeping gene 18SCt.

Western blot Lysates were prepared from cells using RIPA Buffer. Protein samples were resolved on 10% SDS-PAGE and transferred to nitrocellulose membranes (MDI). Membranes were incubated with respective primary antibodies, followed by incubation with secondary HRP-conjugated antibody (Abcam, UK). Chemiluminescence detection reagent (Thermo-Scientific, US) was used to develop blots, and band images were acquired with FluorChem M (Protein Simple, California, USA) followed by quantification using ImageJ analyzer software.

MTT assay 10,000 cells were plated in 96-well plate. Different concentrations of decitabine were added to the cells and kept for 72 h. After incubation, 10 µL of 10 mg/mL MTT was added to each well and incubated at 37 °C for 4 h. 100 µL of Dimethyl sulfoxide (DMSO) was added to each well and absorbance was recorded at 570 nm by an ELISA reader. The control value corresponding to untreated cells was taken as 100%, and the viability of treated cells was expressed as a percentage of the control. Effect of decitabine on enhancing cytotoxicity of chemotherapeutic drugs was observed. For this, two drugs cisplatin and doxorubicin were used and combination experiment with decitabine was performed. Two concentrations of decitabine 1 and 5 µM were used with different concentrations of cisplatin/doxorubicin (1, 3, 5, 7, and 10 µg/mL). Initially, cells were incubated with decitabine for 72 h followed by treatment with cisplatin/doxorubicin for next 72 h. Cells treated with cisplatin and doxorubicin alone were taken as control. MTT was performed as described earlier.

Molecular and Cellular Biochemistry

PI staining and FACS analysis 3 × 105 HT1376 and 1.5 × 105 T24 cells were plated in serum-free media in 6-well plate and incubated with different concentrations of decitabine (1, 5, and 10 µM) for 72 h. Cells were washed and treated with 10 µL of 100 µg/mL ribonuclease. 5 µL PI (from 100 µg/mL stock solution) was added and analyzed by Flow cytometer (FACS Canto II, BD Biosciences, USA). To evaluate the role of decitabine on increasing cytotoxicity of chemotherapeutic drugs cisplatin and doxorubicin to the cancer cells, incubation of cells was done with decitabine (1, 5 µM) for 72 h followed by treatment with cisplatin and doxorubicin (3 µg/mL) next 72 h. Cells were processed as previously described and analyzed by Flow cytometer.

Statistical analysis Data  was expressed as fold change for mRNA expression. Comparsion between treated and control groups were made using the paired student t test. Statistical significance was defined at a p value less than 0.05 (p < 0.05). All data analyses were done with Graph Pad Prism 6 and SPSS 19.0.

Results Status of RASSF1A in bladder cancer cells We assessed expression of RASSF1A in human bladder cancer cells (HT1376 & T24), HeLa cells (Positive control of RASSF1A), and in Healthy control (H.C.) by RT PCR and western blot. We found loss of RASSF1A expression in bladder cancer cells at mRNA as well as at protein levels, whereas RASSF1A expression was present in HeLa and Healthy control (Fig. 1a, b). Further MSPCR revealed methylation of RASSF1A promoter in bladder cancer cells (Fig. 1c). For further confirmation, cells were treated with decitabine (1, 5 µM) and expression of RASSF1A and its promoter methylation were analyzed. Significant increase in RASSF1A expression at mRNA and protein levels was observed due to promoter demethylation after treatment of cells (Fig. 1d–f). Combined results indicate that RASSF1A was inactivated by methylation in bladder cancer cells.

RASSF1A activates hippo pathway and decreases expression of CYR61 and CTGF To identify activation of Hippo pathway, HT1376 and T24 cells were treated with decitabine (1, 5 µM). Phosphorylation status of MST and YAP, major components of the Hippo pathway, was analyzed by Western blot. Levels of

pMST and pYAP were found to be significantly increased (5 µM = p < 0.05) in cells treated with decitabine compared to control, leading to the activation of MST and inactivation of YAP. Further regulation of AREG, CYR61, and CTGF (an oncogenic downstream targets of YAP) by RASSF1A were also observed. Protein levels of CYR61 and CTGF were significantly decreased (5 µM = p < 0.05) in cells after decitabine treatment. Whereas no significant difference was observed for AREG (p > 0.05) after decitabine treatment (Fig. 2).

Effect of decitabine on cell proliferation To investigate the effect of decitabine on cell proliferation and cell cycle in HT1376 and T24 cells, MTT assay and PI staining were conducted, respectively. MTT assay revealed that decitabine significantly inhibited the viability of cells at higher concentration (HT1376: 10 µM-p = 0.03, 15  µM-p = 0.01, 20  µM-p = 0.01; T24: 7  µM-p = 0.04, 10 µM-p = 0.02, 15 µM-p = 0.01, 20 µM-p = 0.01) in both of the cells compared with the untreated controls (Fig. 3a for HT1376 cells and Fig. 3b for T24 cells). Effect of decitabine treatment on cell cycle was analyzed by PI staining using Flow Cytometry. FACS analysis revealed that treatment with the decitabine resulted in cell cycle arrest in G2/M phase in a dose-dependent manner. After dose-dependent treatment, the percentage of diploid cells in G2/M phase progressively increased and the cell percentage in the G0/G1 and S phases was progressively decreased. Representative picture of PI staining of HT1376 cells (Fig. 3c) and bar diagram showing % cells in different phases of cell cycle after treatment with decitabine are shown in Fig. 3d for HT1376 and Fig. 3e for T24.

Decitabine augments cytotoxicity of chemotherapeutic drugs cisplatin and doxorubicin to the bladder cancer cells Two standard drugs cisplatin and doxorubicin were used and combination experiments were performed. MTT assay revealed that cell proliferation in HT1376 and T24 cells treated with combination of decitabine with cisplatin or doxorubicin, showed statistically significant reduced cell proliferation (p < 0.05) at each concentration compared to the cisplatin/doxorubicin-treated cells (Fig. 4). For PI staining, 3 μg/mL cisplatin/doxorubicin concentration was used and combined with either 1 or 5 µM decitabine. Results showed that combination treatment of cisplatin (3 μg/mL) with either 1 or 5 µM decitabine induced more apoptosis (Sub G0/G1 phase) [HT1376 cells (1 μM)Sub G0/G1 = 29.00 ± 1.40; p = 0.015 and T24 cells (1 μM)Sub G0/G1 = 32.4 ± 2.82; p = 0.035 and HT1376 cells (5 μM)-Sub G0/G1 = 35.32 ± 2.29; p = 0.015 and T24 cells

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Fig. 1  Expression of RASSF1A in Bladder cancer cells (HT1376, T24), HeLa, and in Healthy control. a at mRNA level, b at protein level, c RASSF1A methylation status in Bladder cancer cells and in HeLa, d expression of RASSF1A at mRNA level in HT1376 and T24 cells after decitabine treatment (1, 5 µM), e expression of RASSF1A

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at protein level in HT1376 and T24 cells after decitabine treatment (1, 5  µM), f MSPCR of RASSF1A in HT1376 and T24 cells after decitabine treatment (1, 5 µM). ‘*’ a statistically significant difference (p < 0.05). ‘**’ a statistically significant difference (p < 0.001)

Molecular and Cellular Biochemistry

Fig. 2  Effect of decitabine treatment on RASSF1A and hippo pathway molecules. a, b Protein levels of RASSF1A, hippo pathway molecules (MST & YAP), downstream targets (CTGF, CYR61 & AREG) in HT1376 and T24 cells, respectively, after treatment with different

concentrations of decitabine (1, 5 µM) using Western blot. c, d Densitometry analysis of blots using Image J software for HT1376 and T24 cells, respectively. ‘*’ a statistically significant difference (p < 0.05)

(5 μM)-Sub G0/G1 = 41.0 ± 2.54; p = 0.011] in HT1376 and T24 cells, when treated to cisplatin alone (HT1376 cells—Sub G0/G1 = 13.45 ± 2.47 and T24 cells—Sub G0/ G1 = 18.2 ± 2.12) (Fig. 5c, e). Representative picture of combination experiment including decitabine and cisplatin in HT1376 cells is shown in Fig. 5a. Similar results were obtained for combination treatment of doxorubicin with decitabine. Combination treatment either with 1µM

(HT1376 cells-SubG0/G1 = 20.10 ± 2.10; p = 0.047 and T24 Cells—SubG0/G1 = 28.15 ± 1.76; p = 0.027) or 5 μM decitabine (HT1376 cells—SubG0/G1 = 26.40 ± 3.5; p = 0.049 and T24 cells—SubG0/G1 = 35.55 ± 2.33; p = 0.012) induced more apoptosis (Sub G0/G1 phase) in HT1376 and T24 cells, when treated to doxorubicin alone (HT1376 cells—Sub G0/G1 = 10.7 ± 0.94 and T24 cells— Sub G0/G1 = 15.75 ± 2.19) (Fig. 5b, d).

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Molecular and Cellular Biochemistry

Fig. 3  Effect of decitabine treatment on HT1376 and T24 cells proliferation. a MTT assay of HT1376 cells treated with different concentrations of decitabine. b MTT assay of T24 cells treated with different concentrations of decitabine. c Representative PI staining images of HT1376 cells treated with different concentrations of decitabine (1, 5,

10 µM). d For HT1376 and e for T24, bar diagram showing percentage cells in different phases of cell cycle after treatment with different concentrations of decitabine (1, 5, 10 µM). ‘*’ a statistically significant difference (p < 0.05). ‘**’ a statistically significant difference (p < 0.001)

Discussion

drug resistance has not been fully understood. Therefore, this necessitates the identification of genes along with signaling pathways involved in chemo-resistance [23]. RASSF1A is an important tumor suppressor gene and frequent loss of its expression through promoter methylation has been detected in various tumor entities including bladder cancer and correlated with disease severity [10, 24, 25]. In the present study, we found loss of RASSF1A expression in bladder cancer cells (HT1376 & T24) which was associated

Epigenetic modifications play a critical role in the development of cancer by contributing cumulative changes in normal cells undergoing malignant transformation. Genetic changes along with accumulating epigenetic alterations are involved in chemo-resistance in response to therapy [21, 22]. Various molecules and genes have been identified as being involved in drug resistance but actual mechanism underlying

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Fig. 4  Decitabine augments cytotoxicity of cisplatin and doxorubicin to cells (HT1376 & T24). a, b MTT assay of decitabine (1, 5  µM) with cisplatin and doxorubicin, respectively, (1, 3, 5, 7, 10 µg/mL) for

HT1376. c, d MTT assay of decitabine (1, 5 µM) with cisplatin and doxorubicin, respectively, (1, 3, 5, 7, 10 µg/mL) for T24 cells

with hypermethylation of RASSF1A promoter. Further treatment of bladder cancer cells with decitabine leads to RASSF1A restoration supports that RASSF1A expression was drastically reduced due to promoter hypermethylation. This is in concordance with Lee et al. [26] who demonstrated restoration of RASSF1A in bladder cancer cells after treating with decitabine. Hippo pathway is tumor suppressor in nature, controls various functions critical to several carcinogenesis processes such as proliferation, apoptosis, and stem cell maintenance [27]. In activated condition, there is phosphorylation of YAP and TAZ, by Hippo core complex, and thus leads to its retention in the cytoplasm and degradation. While in inactivated state, YAP and TAZ translocate to the nucleus and regulate the activity of various transcription factors involved in cell proliferation, survival, metastasis, and stem cell maintenance [27]. Recently, Sebio and Lenz [28] have described various up stream regulators of hippo pathway such as NF2, KIBRA, and WILLIN complex. Along with this, RASSF1A also acts as an upstream regulator of Hippo pathway. RASSF1A by binding to MST regulates LATS1/2 phosphorylation and affects expression of downstream targets like AREG, survivin, CTGF, and CYR61 [28]. In the present study, restored RASSF1A in cells after treatment with decitabine caused

activation of hippo pathway by activating MST and YAP (pMST & pYAP) followed by decreased expression of oncogenic downstream targets CTGF and CYR61. Hence, RASSF1A decreases expression of downstream targets (CTGF & CYR61) through activation of Hippo pathway in bladder cancer. Although Ahn et al. [29] have previously shown the regulation of AREG by RASSF1A via hippo pathway but in the present study, we could not get significant difference in AREG expression in response to RASSF1A restoration. The reason might be that YAP lacks intrinsic DNA-binding activity, its targets are dictated by its transcription partners in a context-dependent manner [30]. One of the mechanisms by which antineoplastic agents retard tumor growth is by arresting cell cycle progression. Studies have reported antineoplastic potential of decitabine in cholangiocarcinoma and in gastric cancer [31, 32]. In the present study, decitabine treatment significantly suppressed the proliferation of cultured cells and cell cycle arrest at G2/M phase. Our results are in accordance with previous studies and support the fact that decitabine could be proposed as a promising candidate for restricting growth of bladder cancer cells. RASSF1A has been shown to be involved in chemoresistance in solid tumors. Study by Zhao and Dou [13]

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Fig. 5  a Representative PI staining images of HT1376 cells after treatment with combination of decitabine (1, 5  µM) and cisplatin (3 µg/mL). b, c, d, e Bar diagram showing percentage cells in different phase of cell cycle after combination treatment with decitabine (1,

5  µM) with cisplatin or doxorubicin (3  µg/mL) in HT1376 and T24 cells, respectively, ‘*’ a statistically significant difference (p < 0.05). ‘**’ a statistically significant difference (p < 0.001)

reported inactivation of RASSF1A in HCC through hypermethylation, and resistance of cells to anticancer drugs. Similarly, Kassler et al. [14] found loss of RASSF1A expression in ovarian cancer cells through hypermethylation. Treatment with decitabine restored RASSF1A expression, increased taxol sensitivity that suggested the use of epigenetic therapy to overcome taxol resistance in ovarian cancer. Mounting

evidence strongly suggests involvement of hippo pathway molecules YAP [15, 16] and downstream oncogenic targets CTGF and CYR61 [17] in chemotherapeutic drug resistance as well. Demethylating agents have been shown to reduce proliferation, promote apoptosis, and increase sensitivity of cancer cells to chemotherapeutic drugs by reversing hypermethylation of RASSF1A [14, 33].

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Thus, epigenetic regulation of RASSF1A may provide potential target for bladder cancer therapy. We hypothesized that restored RASSF1A by activating hippo pathway leads to decrease expression of CTGF and CYR61 which may enhance cytotoxic effect of chemotherapeutic drugs to bladder cancer cells. To test this hypothesis, we performed combination experiments of decitabine with cisplatin/doxorubicin and found that pretreatment of decitabine enhanced cytotoxicity of drugs. Combination treatment of decitabine and cisplatin/doxorubicin induced significant apoptosis in cells compared to cisplatin/doxorubicin treatment alone. Studies have been carried out to examine the effects of decitabine and chemotherapeutic agents against tumor cells in vitro which supports our results. Plumb et al. [19] found that decitabine sensitizes colon tumors to cisplatin by decreasing hMLH1 promoter methylation. Decitabine was reported to increase the cytotoxicity of cisplatin in lung cancer cells [20]. Similarly, decitabine has been shown to increase sensitivity of renal carcinoma cells against paclitaxel and for bladder cancer cells against cisplatin after combination hence, suggested that combination therapy with decitabine might be a new strategy to improve clinical response of malignancy [18]. For the first time, we have shown that decitabine treatment leads to restoration of RASSF1A, activation of hippo pathway followed by decreased expression of its oncogenic downstream targets leading to augmented cytotoxicity of cisplatin and doxorubicin in bladder cancer. Hence, our data support the potential of RASSF1A as novel therapeutic target through activation of hippo pathway in bladder cancer.

Conclusion RASSF1A-Hippo pathway link may be exploited as an attractive target of decitabine which may further help in augmenting cytotoxicity of chemotherapeutic drugs cisplatin and doxorubicin to bladder cancer cells. Thus, combination treatment of chemotherapeutic drugs along with decitabine may serve as a better therapy for bladder cancer treatment to have better outcome in future. Acknowledgements  Fellowship to M. Khandelwal from Council of Scientific and Industrial Research, India. We acknowledge Dr. P. P. Chattopadhyay (Department of Biochemistry, AIIMS) for providing HeLa cells.

Compliance with ethical standards  Conflict of interest  The authors declare that they have no difference of interest.

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