Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer

ARTICLE IN PRESS Cancer Letters ■■ (2014) ■■–■■

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Original Articles

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Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer Q2 Radha Munagala a,b, Farrukh Aqil a,b, Jeyaprakash Jeyabalan b, Ramesh C. Gupta b,c,* a b

Q3

c

Department of Medicine, University of Louisville, Louisville, KY 40202, USA James Graham Brown Cancer Center, University of Louisville, Louisville, KY 40202, USA Department of Pharmacology and Toxicology, University of Louisville, Louisville, KY 40202, USA

A R T I C L E

I N F O

Article history: Received 4 August 2014 Received in revised form 26 September 2014 Accepted 29 September 2014 Keywords: Cervical cancer Tanshinone IIA HPV E6/E7 oncogenes Tumor suppressor genes Apoptosis p53

A B S T R A C T

Human papilloma virus (HPV) is the well-established etiological factor of cervical cancer. E6 and E7 oncoproteins expressed by HPV are known to inactivate tumor suppressor proteins p53 and pRb, respectively. Tanshinone IIA (Tan IIA) is a diterpenoid naphthoquinone found in the traditional Chinese medicine Danshen (Salvia sp.). Tan IIA has been shown to possess anti-tumor activity against several cancer types. In this study we show that Tan IIA potently inhibited proliferation of the human cervical cancer cells CaSki, SiHa, HeLa and C33a. Mechanistically in HPV positive CaSki cells, Tan IIA was found to (i) downregulate expression of HPV E6 and E7 genes and modulate associated proteins E6AP and E2F1, (ii) cause S phase cell cycle arrest, (iii) induce accumulation of p53 and alter expression of p53-dependent targets, (iv) modulate pRb and related proteins, and (v) cause p53-mediated apoptosis by moderating Bcl2, Bax, caspase-3, and PARP cleavage expressions. In vivo, Tan IIA resulted in over 66% reduction in tumor volume of cervical cancer xenograft in athymic nude mice. Tan IIA treated tumor tissues had lower expression of proliferation marker PCNA and changes in apoptosis targets were in agreement with in vitro studies, further confirming reduced proliferation and involvement of multiple targets behind anticancer effects. This is the first demonstration of Tan IIA to possess significant anti-viral activity by repressing of HPV oncogenes leading to inhibition of cervical cancer. Together, our data suggest that Tan IIA can be exploited as a potent therapeutic agent for the prevention and treatment of cervical and other HPV-related cancers. © 2014 Published by Elsevier Ireland Ltd.

45 Introduction

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Cervical cancer is the second most common life-threatening cancer among women worldwide. Epidemiological and clinical data suggest that infection with high-risk human papilloma virus (HPV), such as 16 and 18, plays a major role in the multi-factorial etiology of cervical cancer [1]. Expression of HPV-specific oncoproteins E6 and E7 is considered essential in initializing and maintaining the malignant growth of cervical cancer. The high-risk HPV E6 protein binds to p53 and stimulates its degradation by an ubiquitindependent protease system, while HPV E7 protein causes destabilization and the disruption of Rb/E2F repressor complexes [2]. Since the growth-regulatory machinery is masked by the expression of HPV E6 and E7 proteins in the carcinoma cells, repression of HPV oncogenes has been exploited as a target to arrest malignant growth in cervical cells [3,4]. The restoration of normal p53 function in cancer cells represents one opportunity that might

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Q1

* Corresponding author. E-mail address: [email protected] (R.C. Gupta).

sensitize cells to cancer therapies or induce apoptosis directly [5–7]. In fact, the possibility of reactivating the p53 pathway has been extensively studied in several cancers [8–12]. Natural products have been identified as promising sources of drugs for cancer prevention and treatment, based on their ability to attack multiple molecular targets [13–17]. Tanshinone IIA (Tan IIA) is a diterpene naphthoquinone from the root of the traditional Chinese medicine, Salvia miltiorrhiza Bunge, reported to possess anti-inflammatory, anti-oxidative and cytotoxic activities [18,19]. It is also known to improve blood circulation and treat chronic hepatitis and hepatic fibrosis [20–22]. Traditional Chinese medicine considers Tan IIA as potential drug for cancer treatment. A recent study demonstrated Tan IIA to cause reversal of the malignant phenotype, decreased migratory and invasive abilities as well as significant increase in the sensitivity of gastric cancer cells to adriamycin and 5-fluro uracil [23]. A few studies have determined growth inhibitory activity of Tan IIA against cervical cancer cells with some insights into molecular targets related to apoptosis [24–26]. However, studies on the HPV oncogenes, one of the most critical factors associated with uterine cervical carcinogenesis, are scanty. Thus, the aim of this study was to determine the antiproliferative activity of Tan IIA against cervical cancer cells with

http://dx.doi.org/10.1016/j.canlet.2014.09.037 0304-3835/© 2014 Published by Elsevier Ireland Ltd.

Please cite this article in press as: Radha Munagala, Farrukh Aqil, Jeyaprakash Jeyabalan, Ramesh C. Gupta, Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer, Cancer Letters (2014), doi: 10.1016/j.canlet.2014.09.037

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special emphasis on viral oncoproteins, and investigate associated molecular mechanism. Here we report that Tan IIA treatment resulted in repression of HPV E6 and E7 oncoproteins, and revival of tumor suppressor proteins p53 and pRb levels, causing downstream modulation of proteins involved in cell proliferation, cell cycle progression, and apoptosis.

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Materials and methods

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Tan IIA was purchased from PhytoMyco, Inc (Greenville, NC). Antibodies against Bax, Bcl2, caspase-3, p53, p21cip1/waf1, p34cdc2, E6, p107, p130, c-myc, E2F1, p-Cdk2 (Thr15), and E6AP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Poly (ADP-ribose) polymerases (PARP), β-catenin, cyclin B1, cyclin D1, cyclin A, cyclin E, Cdc25A, Cdc25B, Cdk2, Bad, Bcl-XL and PCNA were purchased from Cell Signaling (Danvers, MA). Antibody for pRb, p-pRb (Ser608) and E7 were from Abcam (Cambridge, MA), RNA polymerase-related proteins (POLR2A (p), POLR2A, POLR2J) from Assay Biotechnology (Sunnyvale, CA) and β-actin from Sigma-Aldrich (St. Louis, MO). All other chemicals used were of analytical grade.

Chemicals and reagents

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Cell culture

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Human cervical cancer cell lines, CaSki (contains an integrated HPV16 genome as well as sequences related to HPV18; wt p53 and wt pRb), HeLa (HPV 18 +ve; wt p53 and wt pRb), SiHa (HPV16 +ve; wt p53 and wt pRb) and C33a (HPV –ve; mt p53 and mt pRb), and primary human epidermal keratinocytes (HEKn, normal) were obtained from American Type Culture Collection (ATCC, Manassas, VA). The CaSki, HeLa, SiHa and C33a cells were cultured in RPMI 1640 media (Invitrogen, Carlsbad, CA) containing supplements (10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 0.1 mM nonessential amino acids). Primary HEKn cells were cultured in basal cell dermal medium and supplemented with keratinocyte growth kit components and antibiotics (ATCC, Manassas, VA). Cells were maintained in humidified air containing 5% CO2 at 37 °C. Cell lines were authenticated and were negative for mycoplasma.

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Assessment of cell proliferation

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The effect of Tan IIA on cell viability was determined by the MTT assay as describe previously [27]. Cells were treated with Tan IIA (0–25 μM) in the growth medium for 24–72 h.

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Annexin V/propidium iodide (PI) assays for apoptosis by flow cytometry

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For apoptosis assays, cells were stained with annexin V-FITC and PI according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA), and then evaluated for apoptosis by flow cytometry. The apoptotic cells were determined using a BD FACScan flow cytometer (Becton Dickinson, San Jose, CA).

Confocal microscopy

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CaSki cells were seeded in 8-well chamber slides and treated with Tan IIA (5 μM) or vehicle for 24 h. Cells were fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.25% Triton X-100 for 10 min on ice. Cells were blocked with 1% BSA for 30 min, and incubated with primary antibody diluted in 1% BSA for 1 h at room temperature followed by 3 washes with PBS and finally incubated with secondary Alexa Fluor 594-conjugated anti-mouse antibody for HPV-16 E6 and E7 or Alexa Fluor 488-conjugated anti-mouse antibody for p53 or pRb for 1 h at room temperature. After 3 washes with PBS, cells were stained with Phalloidin 488 or 594 for actin filaments and DAPI for detecting nuclei. Cells were mounted and examined under Olympus FluoView FV1000 confocal microscope. Images were captured at 20× magnification and merged with FluoView software.

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Tumor xenograft study

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The animal care and treatments were carried out in strict accordance with the protocol approved by the Institutional Animal Care and Use Committee of the University of Louisville. First, exponentially-growing, CaSki cells were detached by trypsinization, washed, and re-suspended in serum-free RPMI 1640. Cell suspension (5 × 106) was mixed (1:1 volume) with Matrigel (BD Bioscience, Bedford, MA) and injected into the right flank of 5–6 week-old female athymic nu/nu mice (n = 16) (Harlan laboratories, Indianapolis, IN). Tumor bearing mice were randomly divided into two groups with 8 animals each. Animals received either intraperitoneal (i.p.) injection of vehicle or Tan IIA (30 mg/kg in 100 μl) on alternate days for 8 wk. The other group received vehicle only on the same schedule. The mice were weighed weekly to determine any toxicity associated with Tan IIA treatments, and the tumors were measured using digital Vernier calipers. Tumor volume was calculated using the formula: [π/6 × length × width × height]. There was no mortality during the treatment regimen. At the end of the study, animals were euthanized by CO2 asphyxiation, the tumors were harvested, snap-frozen, and stored at −80 °C until analysis.

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Protein extraction and Western blot analysis CaSki cells treated with various concentrations of Tan IIA and tumor tissues from nude mice were lysed in RIPA buffer and protease-inhibitor cocktail (Thermo Scientific, Waltham, MA). The protein concentration of lysate was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). Equal amounts of protein were separated by SDS-PAGE and Western blot analysis as described elsewhere [27].

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Quantitative real-time PCR

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Total cellular RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA) as per manufacturer’s protocol. A high-capacity cDNA reverse-transcriptation kit (Applied Biosystems, Foster City, CA) was used to prepare cDNA. Real-time qPCR was performed using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) on the ABI 7500 Fast real-time PCR system using primers for HPV16 E6, E7 and internal standard TATA binding protein (TBP) [27]. After completion of the RT-PCR, Ct values were obtained from the ABI 7500 fast v2.0.1 software. The ΔΔCt method was used to represent mRNA fold change.

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Analysis of changes in cell morphology

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Changes in cell morphology were studied using DNA-binding dyes, acridine orange (AO) and ethidium bromide (EB), which enable the identification of cells undergoing apoptosis [28]. Briefly, CaSki cells were treated with either vehicle (DMSO) or Tan IIA at 2.5–10 μM for 24 and 48 h. One μl of dye mixture (100 μg/ml AO and 100 μg/ ml EB in distilled water) was mixed with 9 μl of cell suspension (0.5 × 106 cells/ml) on a clean microscope slide and immediately examined by fluorescence microscopy (NIKON, Melville, NY) at 100× magnification. In addition, cells were examined for nuclear morphology of apoptosis (chromatin condensation, DNA fragmentation) by labeling with DAPI (Sigma, St. Louis, MO).

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Cell cycle analysis by flow cytometry

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CaSki cells (1 × 106) treated with Tan IIA were harvested by trypsinization, followed by centrifugation (300 × g for 5 min at 4 °C) and washed twice with ice-cold PBS. The cells were then fixed in ice-cold 70% ethanol for 30 min at 4 °C, centrifuged again and washed twice with PBS. After removal of the supernatant, the cells were resuspended in 1 ml of DNA-staining solution (20 μg/ml of PI and 100 μg/ml of RNase A in PBS) and incubated for 30 min at room temperature. DNA content was analyzed by flow cytometry. The population of cells in each cell cycle phase was determined using FlowJo software v7.2.5 (Treestar, Ashland, OR).

Statistical analysis

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For both the in vitro and in vivo studies, the statistical significance was evaluated by the two-tailed Student’s t-test. P-values <0.05 were considered to be statistically significant. The data points shown in the figures represent the mean ± SD. In vitro assessments were performed in three independent experiments to confirm the results.

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Results Tan IIA exhibits antiproliferative activity, and induces morphological changes and apoptosis in CaSki cells Tan IIA inhibited the growth of cervical cancer cell lines CaSki, SiHa HeLa and C33a cells in a dose-dependent manner after 24 h treatment. The proliferation of primary epidermal keratinocytes was only slightly affected by Tan IIA at the similar concentration (Fig. 1a). All HPV-positive cell lines, CaSki, SiHa and HeLa exhibited a decrease in the levels of E6 and E7 oncoproteins with Tan IIA treatment.

Please cite this article in press as: Radha Munagala, Farrukh Aqil, Jeyaprakash Jeyabalan, Ramesh C. Gupta, Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer, Cancer Letters (2014), doi: 10.1016/j.canlet.2014.09.037

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Fig. 1. Tan IIA inhibits cervical cancer cell proliferation. (a) Data represent dose-dependent viability analyzed by MTT assay in the CaSki, SiHa, HeLa and C33a human cervical cancer cell lines and primary epidermal keratinocytes (HEKn) after 48 h of treatment. (b) Western blot of cell lysates prepared after treating cervical cancer cells with 0–10 μM Tan IIA for 24 h and probed for E6, E7, p53 and pRb proteins. (c) Time-dependent anti-proliferative effects of Tan IIA on CaSki cells. (d) Representative images of morphological changes in CaSki cell treated with 5 μM Tan IIA for 24 h are shown. Left: ethidium bromide (EB) and acridine orange (AO) staining, solid white arrow indicates membrane blebbing and dashed yellow arrow indicates apoptotic cell. Right: DAPI staining for changes in nuclear morphology, dashed white arrows indicate chromatin condensation and fragmented nuclei. (e) Dose- and time-dependent effect of Tan IIA on apoptosis. Percent of early, late apoptosis and live cells at indicated dose of Tan IIA. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Radha Munagala, Farrukh Aqil, Jeyaprakash Jeyabalan, Ramesh C. Gupta, Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer, Cancer Letters (2014), doi: 10.1016/j.canlet.2014.09.037

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As E6 and E7 oncoproteins are known to cause degradation of p53 and pRb, respectively, it would be expected that downregulation of E6 and E7 oncogenes result in the restoration of p53 and pRb levels. In conformation toward this end we observed increased expression of p53 in all the HPV-positive cell lines and while increase in pRb levels was much evident with CaSki cells, this was not clear with HeLa and SiHa. In contrast, HPV-negative and mutant p53 and pRb-expressing C33a cells had no significant effect of Tan IIA on p53 or pRb suggesting a p53-independent mechanism behind the apoptosis (Fig. 1b). Although Tan IIA was equally effective against the all three HPV-positive cervical cell lines tested, we chose to perform detailed mechanistic studies with CaSki as it carried integrated HPV 16 genome as well as sequences related to HPV-18 and exhibited highest derepression of both p53 and pRb tumor suppressor proteins with Tan IIA treatment compared to other HPV-positive cell lines tested. Time-dependent effect of Tan IIA treatment on CaSki cells is shown in Fig. 1c. Tan IIA-treated CaSki cells elicited morphological changes such as membrane blebbing, chromatin condensation, DNA fragmentation and the manifestation of early and late apoptosis (Fig. 1d). Apoptosis was further confirmed by annexin V-FITC and PI-staining, where CaSki cells treated with Tan IIA resulted in a significant dose- and time-dependent induction of apoptosis. In comparison with vehicle treatment, a significant

increase in early-apoptotic cells was observed at concentrations of 2.5–10 μM Tan IIA after 24 h treatment (Fig. 1e). A shift from early-apoptotic to late-apoptotic cell population was observed after 48 h (Fig. 1e). These results confirm the induction of apoptosis as a major mechanism behind Tan IIA antiproliferative activity.

35 Tan IIA repressed HPV16-E6 /E7 oncoproteins The effect of Tan IIA treatment on HPV16 E6 and E7 oncogenes in CaSki cells was investigated by real-time qPCR. Our results indicate that Tan IIA treatment (2.5–10 μM) caused significant (P ≤ 0.0001; ANOVA) downregulation of both HPV16 E6 and E7 transcripts, and the effect was dose dependent. A reduction of 7 and 10-fold in the mRNA expression of HPV16 E6 and E7, respectively, was observed with 10 μM Tan IIA (Fig. 2a). Western blot analysis confirmed these results, as reflected by decrease in HPV16 E6 and E7 protein levels. E6-associated E3 ubiquitin ligase E6AP that targets p53 degradation and E7-associated transcription factor E2F1 were also decreased with Tan IIA (Fig. 2b). These observations confirm the ability of Tan IIA to cause repression of HPV oncogenes.

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Relative mRNA expression

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Fig. 2. Tan IIA represses HPV oncogenes. (a) CaSki cells were treated with vehicle or Tan IIA for 48 h and HPV16 E6 and E7 mRNA levels were measured by qPCR. Data represent mean ± SD of relative mRNA expression to vehicle treated cells. ***P < 0.001. (b) Western blot analysis of HPV oncogenes and associated proteins from Tan IIA-treated CaSki cells at indicated doses after 48 h of treatment (left). Representative blots are shown. Bar graph (right) shows the relative densitometry intensity of bolts normalized to β-actin using Image J software.

Please cite this article in press as: Radha Munagala, Farrukh Aqil, Jeyaprakash Jeyabalan, Ramesh C. Gupta, Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer, Cancer Letters (2014), doi: 10.1016/j.canlet.2014.09.037

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Tan IIA restored p53, pRb and associated protein levels Tan IIA increased p53 levels dose dependently, and this effect further resulted in modulation of downstream targets such as, p21cip1/waf1, p34 and c-myc levels (Fig. 3a). Our results indicated substantial increase of p53-responsive p21cip1/waf1 levels and reduction in p34 cdc2 and c-myc levels. pRb and its associated proteins including p107 and p130 were induced dose-dependently by Tan IIA (Fig. 3b). Downregulation of the viral oncoprotein E6 and E7 expression and increase in tumor suppressor proteins p53 and pRb expression by Tan IIA was also confirmed by confocal microscopy (Fig. 3c). These results establish that Tan IIA likely causes the restoration of the vital tumor suppressor genes p53 and pRb to elicit anti-cancer effects in CaSki cancer cells.

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Tan IIA induces S phase arrest by modulating proteins involved in cell cycle progression To determine the effect of Tan IIA on cell cycle progression, we performed cell cycle analysis after treating CaSki cells with Tan IIA for 12–72 h (Fig. 4a). Our results showed a significant alteration in cell cycle progression; in particular, the S phase of cell cycle progression was arrested compared to vehicle treatment. We further investigated its effect on the cell cycle regulators. We found Cdc25A and Cdk2 levels to decrease with increase in pCDK2 in response to Tan IIA, while no significant changes in the protein levels of Cdc25B was noted. Activation of Cdk2 by interaction with cyclin E in late G1 phase and cyclin A in S phase determines cells to enter into and progress through S phase [29]. We observed decrease in both cyclin A and cyclin E with Tan IIA treatment at 48 h compared to control cells. In addition, cyclin B1, cyclin D1 and β-catenin expression levels were also found to decrease. We noted Tan IIA to decrease pRb phosphorylation which is the target of Cdk2. In the absence of Cdc25A, Cdk2–cyclin E/A complex is known to remain in the inactive hyperphosphorylated form and induces S-phase arrest [29,30].

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Tan IIA affects RNA polymerase IIA activity Tan IIA resulted in a decrease in the protein levels of major subunit of RNA polymerase IIA, POLR2A and its phosphorylation form, POLR2A(p) in a dose-dependent manner. We also observed a dose-dependent decrease in the expression levels of another RNAPII subunit protein POLR2J (Fig. 5a) with Tan IIA treatment. These results suggest that Tan IIA cytotoxic effects are partly induced by effecting RNA polymerase activity required for translation of proteins thus inhibiting cancer cell survival.

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Tan IIA caused caspase-dependent apoptosis We investigated the modulation of p53-dependent apoptotic pathway molecules by Tan IIA. Tan IIA treatment resulted in a decrease in the anti-apoptotic protein Bcl2, Bcl-XL and an increase in pro-apoptotic protein Bax and Bad levels. The Bax/Bcl2 ratio was calculated by quantitative densitometry of Bax and Bcl2 protein expression levels. Bax/Bcl2 ratios of 3.3, 8.8 and 5.9 were observed with 2.5, 5 and 10 μM Tan IIA respectively, indicating susceptibility of CaSki cells for apoptosis with Tan IIA. p53 is also known to regulate the activation of caspase-3 to induce apoptosis. Thus, as expected, we observed reduced pro-caspase-3 levels with increase in cleaved caspase-3 in a dose-dependent manner and also increase in cleaved PARP was also observed (Fig. 5b). These results suggest that Tan IIA regulated and induced apoptosis in a caspasemediated manner.

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Tan IIA inhibits growth of subcutaneous xenograft tumor in nude mice Treatment with Tan IIA (30 mg/kg) on alternate days for 8 weeks caused a significant inhibition of CaSki tumor xenograft growth in nude mice compared to control (90 ± 66 versus 267 ± 100 mm3; p ≤ 0.01) (Fig. 6a). We did not observe any gross signs of toxicity and/ or possible side effects/mortality during the Tan IIA treatment duration, as reflected by unaltered body weights, equal diet consumption and the animal movement compared with the control group. A decrease in HPV E6 and E7 expression levels with a significant increase in p53, p21cip1/waf1, pRb and p130 levels was observed in tumor tissues treated with Tan IIA, this was in agreement in vitro studies (Fig. 6b). Proliferation marker (PCNA) levels were significantly reduced in the tumors of Tan IIA treated animals compared to vehicle treated group indicating inhibition of tumor proliferation behind the observed anti-tumor effects. Increase in proapoptotic protein Bax and decrease in anti-apoptotic Bcl2 (Fig. 6b) in Tan IIA treated animals further supported the involvement of apoptotic pathways behind antitumor effects. Discussion The main objective of the present study was to evaluate anticancer efficacy and associated mechanisms of action of Tan IIA in human cervical cancer cells, both in cell culture and in vivo. In vitro treatment with Tan IIA markedly inhibited cervical cancer cell proliferation in HPV-positive, CaSki, HeLa, SiHa and HPV-negative C33a cells. HPV E6 and E7 oncoproteins exert profound effects on tumor suppressor proteins p53 and pRb respectively, by accelerating their ubiquitin-mediated degradation [31,32]. Most primary cervical cancers and cancer cell lines are known to harbor wild-type p53 and p105Rb genes [33,34]. Therefore, it is presumed that agents with a capability of repressing HPV oncogenes would cause reactivation of tumor suppressor pathways, and have potential therapeutic implications in treating cervical cancers. To this end, we demonstrated that Tan IIA downregulated expression levels of oncogenes E6 and E7 in all the HPV-harboring cell lines. We further questioned if repression of HPV oncogenes by Tan IIA caused reactivation of dormant tumor suppressor pathways. Our results confirmed that repression of E6 translated into restoration of p53 in all HPVpositive cells. Although marked decrease in E7 expression was observed with Tan IIA in all HPV-positive cells, much pronounced restoration of pRb levels was observed only in CaSki cells but not in SiHa and HeLa cells. As per earlier reports, it is possible that inhibition of E7 results in a dephosphorylation of pRb, without an increase in overall pRb levels [35]. While HPV E6 and E7 can immortalize cells independently, it is noted that the co-operative interactions between E7, pRb and E2F transcription factors substantially enhance immortalization efficiency. E7 binding to pRb results in hyper-phosphorylation of pRb and release of E2F transcription factors which activate genes for cell proliferation [35,36]. Our findings suggested that Tan IIA downregulated both E7 and the major transcription factor E2F1 in CaSki cells. Although Tan IIA caused significant growth inhibition of HPVnegative C33a cells, a change in either p53 or pRb levels was not observed. This is somewhat expected since C33a contains mutated p53 and pRb protein with a longer half-life, which induces its nuclear accumulation [33]. In agreement with our observation, changes in p53 levels in C33a cells were not observed upon treatment with several genotoxic agents [37]. Furthermore, C33a carries a mutation in residue 273, which is not included within the apoptotic domains. In this respect, it has been shown that, p53 can induce apoptosis independently of its transactivation function [38,39]. Thus, in case of C33a cells Tan IIA could be induce apoptosis via alternative mechanisms such as p53-independent caspase activation.

Please cite this article in press as: Radha Munagala, Farrukh Aqil, Jeyaprakash Jeyabalan, Ramesh C. Gupta, Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer, Cancer Letters (2014), doi: 10.1016/j.canlet.2014.09.037

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Fig. 3. Tan IIA modulates tumor suppressor p53 and pRb proteins. CaSki cells were treated with vehicle or Tan IIA at indicated doses for 48 h. (a) Expression levels of p53 and p53-associated proteins (left) and relative densitometry intensity of bolts normalized to β-actin (right). (b) Expression levels of pRb and related protein (left) and relative densitometry intensity of bolts normalized to β-actin (right). Representative blots are shown. (c) Confocal photomicrographs (20×) of representative CaSki cells showing localization of p53, pRb, HPV E6 and E7 proteins. Cells were treated with vehicle (left) and Tan IIA (5 μM) (right) are shown. Alexa Fluor 594-conjugated antibody for HPV16 E6 and E7 (red) or Alexa Fluor 488-conjugated antibody for p53 and pRb (green) was used to detect localization of these proteins (first lane). Phalloidin 488 (green) or 594 (red) was used to visualize actin filaments (second lane) and DAPI was used to visualize DNA (third lane). Overlay (merge) of the all the 3 images is shown in lane four. The scale represents a length of 100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article in press as: Radha Munagala, Farrukh Aqil, Jeyaprakash Jeyabalan, Ramesh C. Gupta, Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer, Cancer Letters (2014), doi: 10.1016/j.canlet.2014.09.037

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Fig. 4. Tan IIA induces S-phase cell cycle arrest and modulates related proteins. (a) CaSki cells were treated with either vehicle or Tan IIA at indicated doses for 12–72 h and cell cycle arrest was measured by flow cytometry. (b) Western blot analysis of p-Cdk2, Cdk2, p-pRb Cdc25A and Cdc25B proteins at 48 h of Tan IIA treatment (left) and relative densitometry intensity of blots normalized to β-actin (right). (c) Western blot analysis of cyclin proteins at 48 h of Tan IIA treatment (left) and relative densitometry intensity of blots normalized to β-actin (right). Representative blots are shown.

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Our results further indicated Tan IIA to cause cell cycle arrest at S phase, with modulation of the major cell cycle regulators such as p34cdc2, p21cip1/waf1, cyclin A, cyclin E, cdc25A and Cdk2. During S phase, Cdc25A is involved in activating cyclin E-Cdk2 and cyclin A-Cdk2 complexes, which are required for initiation of DNA replication and G1/S transition [29,40]. We observed a dramatic decrease in the expression of Cdc25A and Cdk2 proteins in Tan IIA treated cells, leading to reduction of Cdc25A phosphatase activity, which was also evidenced by the increase of inhibitory phosphorylated p-Cdk2 (Tyr15). Rb protein being direct substrate of Cdk2 kinase,

a decrease in Rb phosphorylation was observed in Tan IIA treated cells and seemed to be consistent with the Cdk2 inhibition [29,40]. We further showed that this cell cycle arrest was accompanied by a decrease of cyclins A and E protein levels from the Tan IIA treatment and increase in p21cip1/waf1 that acts as universal inhibitor of Cdks [41]. Downregulation of Cdc25A with subsequent inhibition of Cdk2 activity is one of the possible mechanisms by means of which cells may slow S-phase [29,30]. β-catenin is involved in cyclin D1 transcriptional regulation [42] and has been shown to accelerate HPV-16 mediated cervical

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Fig. 5. Tan IIA perturbs RNA polymerase-related proteins and modulates apoptosis-associated proteins. CaSki cells were treated with vehicle or Tan IIA at indicated doses for 48 h and cell lysates were used for Western blot analysis and probed for (a) RNA polymerase related proteins and (b) apoptosis-related proteins (left). Bar graph shows the relative densitometry intensity of blots normalized to β-actin (right). Representative blots are shown.

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carcinogenesis [43]. We noted that Tan IIA caused downregulation of β-catenin, cyclin D1 and cyclin B1 levels. Our findings are in agreement with Joe et al. [44] who observed cyclin D1 to be the initial cyclin affected during resveratrol induced S-phase arrest. The catalytic subunit of p34cdc2 and the regulatory subunit of cyclin B1 complex to form an active heterodiamer called ‘mitosis-promoting factor’ [45]; thus, a decrease in cyclin B1 and p34cdc2 expression levels by Tan IIA could further inhibit G2-phase entry. Taken together, these findings indicate that Cdc25A–Cdk2–cyclin E/A axis along with decrease in p34cdc2, cyclin B1 and cyclin DI to be involved in Tan IIA induced S phase arrest. Zhang et al. [46] had earlier suggested DNA-conformationaldamage-dependent RNAPII response up on groove binding to be the molecular basis of the antitumor property of Tan IIA. In this study, we examined RNAPII-associated proteins and found that Tan IIA treatment caused a significant decrease in the largest subunit POLR2A as well as the phosphorylated form of this subunit POLR2A (p). It is known that phosphorylation of POLR2A by CDK7 is associated with transcription initiation. Other RNAPII subunit protein POLR2J was also decreased although the exact regulation of these proteins by Tan IIA is poorly understood. Tan IIA was shown to cause DNA structure damage resulting in the inhibition of RNAPII binding to DNA and the initiation of RNAPII phosphorylation, or complete phosphorylation and degradation of RNAPII followed by p53 activation and apoptosis [46]. p53 can induce apoptosis either by a transcription-dependent or a transcription-independent mechanism. Studies have demonstrated the direct activation of the pro-apoptotic protein Bax to

accumulate in mitochondria in response to death signals in p53mediated apoptosis [47]. We confirmed that Tan IIA-induced cell death in CaSki cells was accompanied by up-regulation of Bax and Bad and downregulation of the anti-apoptotic protein Bcl2 and BclXL proteins. Activation of procaspase-3 caused by cleavage, caspase-3 results in the activated form of caspase-3 subsequently leads to PARP cleavage [48], a hallmark of apoptosis. Previous studies have dem- Q4 onstrated that only cancer cells harboring wt-p53 but not mutant could induce caspase-dependent, p53-mediated apoptosis [49,50]. Thus, Tan IIA treatment of CaSki cells clearly demonstrated the restoration of wild type p53 levels, resulting in the modulation of the Bax/Bcl2 ratio and caspase-3 mediated apoptosis. Based on the relatively high antiproliferative activity of Tan IIA against cervical cancer cells in vitro, we further tested its efficacy in a pre-clinical study with subcutaneous xenografts in athymic nude mice. In this study, the animals treated with Tan IIA (30 mg/kg) for 8 weeks exhibited a significant reduction in tumor volume (nearly 66%), with no apparent toxicity. Our data demonstrated that Tan IIA has a strong and significant in vivo antiproliferative effect as reflected by lower PCNA levels in Tan IIA-treated animals compared to vehicle treatment. We also demonstrated that the in vivo protein markers in the tumors treated with Tan IIA were modulated in similar trends as observed in in vitro analysis, confirming the mechanism of action of Tan IIA. As noted in the introduction we examined the anticancer effects of Tan IIA with special emphasis on understanding its effects of viral oncogenes E6 and E7. Our findings that Tan IIA treatment causes suppression of HPV E6 and E7 oncogene expression consequently

Please cite this article in press as: Radha Munagala, Farrukh Aqil, Jeyaprakash Jeyabalan, Ramesh C. Gupta, Tanshinone IIA inhibits viral oncogene expression leading to apoptosis and inhibition of cervical cancer, Cancer Letters (2014), doi: 10.1016/j.canlet.2014.09.037

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Fig. 6. Antitumor activity of Tan IIA on human cervical tumor xenografts in nude mice. (a) CaSki cells (5 × 106) were injected subcutaneously in athymic nude mice (n = 8). Tan IIA (30 mg/kg) or vehicle was given i.p., on alternate days and tumor volume measured once a week. Data represent mean ± SD; (*P < 0.05;**P < 0.01;***P < 0.001, t-test). (b) Western blot analysis of indicated proteins in tumor tissue of representative animals (n = 3) treated with either vehicle or Tan IIA (30 mg/kg) (left). Relative densitometry intensity of bolts normalized to β-actin (right) is shown.

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resulting in revival of p53 and pRb levels are novel and could bear great importance for all HPV-associated human cancers, including cervical cancer. Anti-cancer effects were initiated in a p53-dependent manner through activation of its downstream responsive genes, including the p21cip1/waf1, cell cycle arrest in S phase, Bax and caspase-3 activation. Recent findings by Pan et al. [26] indicated Tan IIA exhibited strong growth inhibitory effects in CaSki cervical cancer cells

via a different molecular axis by upregulation of the p38 phosphorylation and JNK signaling. Their proteomics data further indicated activation of ER stress pathways to cause apoptotic cell death. Taken together, these observations indicate that the mechanism of action of Tan IIA is by attacking multiple molecular targets and by moderation of more than a single signaling pathway. This could be beneficial in achieving greater treatment efficacy.

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In summary, our results show that Tan IIA has profound in vitro and in vivo antiproliferative activities against cervical cancer. This is the first demonstration of the ability of Tan IIA to repress HPV E6 and E7 oncogenes, resulting in reactivation of p53-dependent tumor suppressor activity leading to growth inhibition of cervical cancer cells. Our findings provide a strong basis for the further exploration of Tan IIA as a therapeutic drug against cervical and other HPV-related cancers, either alone or adjuvant to standard chemotherapeutic agents.

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Q5 Funding Agnes Brown Duggan Endowment, Hemsley Funds and the James Graham Brown Cancer Center. R.C.G. holds the Agnes Brown Duggan Chair in Oncological Research. Conflict of interest The authors declare that they have no competing interests.

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