Sildenafil increases chemotherapeutic efficacy of in and ameliorates cardiac dysfunction

Anindita Dasa,1, David Durranta, Clint Mitchellb, Eric Maytona, Nicholas N. Hokea, Fadi N. Sallouma, Margaret A. Parkb, Ian Qureshia, Ray Leeb, Paul Dentb, and Rakesh C. Kukrejaa,1

aDivision of Cardiology, Department of Internal Medicine, Virginia Commonwealth University Pauley Heart Center and bDepartment of Biochemistry and Molecular Biology, Virginia Commonwealth University Medical Center, Richmond, VA 23298

Edited* by Herbert Weissbach, Florida Atlantic University, Boca Raton, FL, and approved August 17, 2010 (received for review May 25, 2010) We have shown that the potent phosphodiesterase-5 (PDE-5) enocarcinoma, bladder squamous carcinoma, and lung cancers inhibitor sildenafil (Viagra) induces a powerful effect on reduction (15–17), suggesting its potential role in controlling tumor cell of infarct size following ischemia/reperfusion injury and improve- growth and death. Sildenafil and vardenafil suppress tumor cell ment of left ventricular dysfunction in the failing heart after growth and induce caspase-dependent apoptosis in B-cell chronic myocardial infarction or doxorubicin (DOX) treatment. In the present lymphatic leukemia (18, 19). Another PDE-5 inhibitor, exisulind study, we further investigated the potential effects of sildenafilon ( sulfone), and its higher affinity analogs also induce ap- improving antitumor efficacy of DOX in prostate cancer. Cotreat- optosis and inhibit cell proliferation in colon tumor cells lines by ment with sildenafil enhanced DOX-induced apoptosis in PC-3 and activating PKG and phosphorylation of β-catenin (20). These DU145 prostate cancer cells, which was mediated by enhanced gen- compounds also inhibit growth and induce apoptosis in several eration of reactive oxygen species, up-regulation of caspase-3 and human prostate cancer cell lines and prostate cancer xenografts in caspase-9 activities, reduced expression of Bcl-xL, and phosphoryla- nude mice (21–23). A low dose combination of colecoxib, tion of Bad. Overexpression of Bcl-xL or dominant negative caspase a cyclooxygenase-2 (COX-2) inhibitor, with exisulind prevents 9 attenuated the synergistic effect of sildenafil and DOX on prostate prostate carcinogenesis by altering key molecular events (24). cancer cell killing. Furthermore, treatment with sildenafil and DOX in In the present study, we tested whether sildenafil potentiates PHARMACOLOGY mice bearing prostate tumor xenografts resulted in significant in- the antitumor efficacy of DOX in prostate cancer. Our results hibition of tumor growth. The reduced tumor size was associated show that DOX and sildenafil induce a potent antitumor effect with amplified apoptotic cell death and increased expression of ac- in prostate cancer while simultaneously providing a cardiopro- tivated caspase 3. Doppler echocardiography showed that sildenafil tective effect. treatment ameliorated DOX-induced left ventricular dysfunction. In conclusion, these results provide provocative evidence that sildena- Results fil is both a powerful sensitizer of DOX-induced killing of prostate Sildenafil Potentiates DOX-Induced Killing of Prostate Cancer Cells. cancer while providing concurrent cardioprotective benefit. First, we examined the dose-dependent effect of DOX treatment in PC-3 and DU145 cells. Cell growth was reduced in a dose-de- apoptosis | phosphodiesterase-5 | reactive oxygen species pendent manner with DOX in both cells (Fig. 1 A and B). Cotreatment with sildenafil resulted in an additive effect on DOX- rostate cancer remains among the most frequently diagnosed induced reduction of proliferation (Fig. 1 A and B). Cell killing Psolid tumors in men and is one of the leading causes of cancer- assessed by trypan blue exclusion assay also confirmed similar ad- related deaths in Western countries (1). Treatment options are ditive effect (Fig. 1 C and D). DOX treatment also increased ap- limited and are associated with significant morbidity and mortality. optosis as evaluated by TUNEL assays (Fig. 1 E and F). The Doxorubicin [(DOX) Adriamycin] is a broad-spectrum antitumor sildenafil and DOX combination further enhanced apoptosis in PC- antibiotic that has been widely used for treatment of several can- 3 and DU145 cells, whereas sildenafil alone had no effect. Colony cers, including breast, ovarian, and prostate cancers (2). The ef- formation assays performed using median dose effect isobologram fectiveness of DOX is limited due to its high toxicity and side effects, analysis further corroborated the synergistic effect of sildenafiland including myelosuppression, alopecia, acute nausea, vomiting, DOX in enhancing cell killing (Fig. 2A). In contrast, DOX treat- stomatitis, cumulative cardiotoxicity (3), and strong multidrug re- ment induced cell death in the normal prostate epithelial cells sistance response in tumor cells after repeated administration (4). (PrEC), which was significantly reduced by sildenafil (Fig. 2B). Sildenafil (Viagra), vardenafil (Levitra), and tadalafil (Cialis) Sildenafil treatment alone had no effect on cell death in the PrEC or inhibit phosphodiesterase-5 (PDE-5), the predominant enzyme in prostate cancer cells. the corpus cavernosum that plays an essential role in vascular Apoptosis was also quantified using Annexin-V-FITC and smooth muscle contraction through specific regulation of cGMP propidium iodide (PI) staining followed by flow cytometry anal- (5). We have demonstrated that sildenafil and other PDE-5 ysis. The cells in the subpopulations labeled by staining of inhibitors induce a powerful protective effect against ischemia/ Annexin-V-FITC(+)/PI(−) were indicative of early apoptotic reperfusion injury (6–10), DOX-induced cardiomyopathy (11), and myocardial infarction-induced heart failure (12). The cardiopro- tective effect is attributed to limiting apoptosis and necrosis Author contributions: A.D. and R.C.K. designed research; A.D., D.D., C.M., E.M., N.N.H., through several mechanisms. These include enhanced expression F.N.S., M.A.P., and I.Q. performed research; R.L. and P.D. contributed new reagents/ana- of nitric oxide synthase (NOS), particularly the endothelial NOS lytic tools.; A.D. and D.D. analyzed data; and A.D. and R.C.K. wrote the paper. (eNOS) and inducible NOS (iNOS); activation of protein kinase C The authors declare no conflict of interest. and protein kinase G (PKG); phosphorylation of ERK1/2; PKG- *This Direct Submission article had a prearranged editor. dependent phosphorylation of GSK-3β; up-regulation of Bcl-2/ Freely available online through the PNAS open access option. Bax; and opening of the mitochondrial KATP channels (6–9, 12–14). 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. It has been shown that PDE-5 expression is increased in multiple This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. human carcinomas including metastatic breast cancers, colon ad- 1073/pnas.1006965107/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1006965107 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 A B 120 120 DOX DOX 110 DOX+ Sild 100 DOX+Sild 100 * 80 90 60 80 * * 70 40 Viable cells (% Control) of Viable cells *

(% Control) of 60 * * 20 * * 50 0 40 0.00 0.25 0.50 0.75 1.00 0.00 0.25 0.50 0.75 1.00 1.25 1.50 DOX ( M) DOX ( M)

C D 35 45 * * 40 30 35 25 30 25 20 * 15 20 * 15 10 Cell Death (%)Cell Death Cell(%) Death 10 5 5 0 0 l o X d ld O il Sild ntr D S DOX X+Si Control Co O D DOX+Sild Fig. 1. Sildenafil (Sild) enhances DOX-induced cell death. Viability of (A) PC-3 and (B) DU145 cells after 72 h of treat- ment with different concentrations of DOX and/or Sild E F (10 μM). Red line represents DOX only; green line represents 40 70 DOX + Sild (10 μM) (*P < 0.001 vs. respective concentration of 60 30 * DOX; n = 6). Cell death assessed by trypan blue exclusion 50 * assay after 24 h of treatment of (C) PC-3 with 1.5 μM DOX ± 40 * 20 μ μ ± μ < * 30 10 M Sild and (D) DU145 with 0.5 M DOX 10 M Sild (*P α 20 0.001 vs. control and P < 0.001 vs. DOX; n = 6). Sild enhances 10 (% of Cells) Total (% of Total Cells) 10

TUNEL PositiveTUNEL Cells DOX-induced apoptosis. Percentage of TUNEL-positive nuclei TUNEL PositiveCells 0 0 in (E) PC-3 cells after 72 h of treatment with 1.5 μM DOX ± ol X ld l X d O ro ild il ntr D Sild μ t S 10 M Sild and (F) DU145 cells after 72 h of treatment with n DO +S Co α Co X DOX+Si μ ± μ < < DO 0.5 M DOX 10 M Sild (*P 0.001 vs. control and P 0.001 vs. DOX; n = 3). Results are presented as means ± SE.

cells, whereas those labeled by Annexin-V-FITC(+)/PI(+) were cotreatment with both sildenafil and DOX in PC-3 and DU145 indicative of late apoptotic/necrotic cells. DOX induced apoptosis cells (Fig. 5A). Bcl-2 expression was diminished in DU145 cells after 72 h of treatments in PC-3 (7.52%) and DU145 cells but remained unaltered in PC-3 cells following treatment with (45.01%) compared with control (5.49% in PC-3 and 5.52% in sildenafil and DOX (Fig. 5B and Fig. S4A). Similarly, the ex- DU145) (Fig. S1 A and B). Cotreatment with sildenafil and DOX pression of the antiapoptotic protein Bcl-xL was reduced with increased apoptosis relative to DOX alone in both cell lines sildenafil and DOX compared with individual treatments or (18.71% in PC-3 and 56.82% in DU145 cells) (Fig. S1 A and B). control in both cell lines (Fig. S4B). Bad belongs to the proa- Moreover, the combination treatment increased apoptotic cell poptotic members of the Bcl-2 family and forms a complex with death in other cancer cell types including sarcoma OSAC-1 (Fig. Bcl-xL thereby preventing its antiapoptotic effects. Phosphoryla- C D E S1 ), ovarian cancers UCI 101 (Fig. S1 ), and A2780 (Fig. S1 ). tion of Bad impairs its binding to Bcl-xL and therefore abrogates Bad’s proapoptotic effects (27). DOX reduced Bad phosphory- Sildenafil Enhances DOX-Induced Generation of Reactive Oxygen lation and sildenafil and DOX further decreased Bad phosphor- Species. Reactive oxygen species (ROS) generation is the key ylation (Fig. 5B and Fig. S4C). DOX induced the proapoptotic component of antitumor activity of in a variety of protein Bax, which was further enhanced by cotreatement with tumor cells (25, 26). We tested whether sildenafil enhances cell sildenafil in PC-3 cells (Fig. 5B and Fig. S4D). Overexpression of killing through increased ROS generation that was measured by fi exposing DOX- and/or sildenafil-treated cells to the indicator dye Bcl-xL inhibited cell death with sildena l and DOX compared fl with DOX alone (Fig. 5D). dichlorodihydro uorescein diacetate (H2DCFDA). As expected, fi DOX increased ROS levels in PC-3 and DU145 cells as indicated Caspase-9 activity was unchanged with sildena l although it in- C by positively stained cells (H DCFDA green fluorescence) (Fig. 3 creased after DOX treatment (Fig. 5 ). Caspase-9 activity was fur- 2 fi A and B, and Fig. S2 A and B). However, cells exposed to sildenafil ther increased with sildena l and DOX treatment compared and DOX further boosted ROS generation. In contrast, the sil- with DOX alone. Overexpression of dominant negative caspase 9 denafil and DOX combination attenuated DOX-induced ROS (dnCasp9) attenuated the synergistic effect of sildenafil and DOX on generation in the PrEC normal cells (Fig. 4 A and B). Sildenafil cell death compared with cells infected with empty vector (Fig. 5E). alone had no effect on ROS generation in normal or cancer cells. fi Furthermore, incubation with a putative antioxidant, mercapto- Sildena l Potentiates DOX-Induced Inhibition of Prostate Tumor fl propionyl glycine (MPG), attenuated the enhanced killing effect Xenograft Growth. Treatment of nude mice carrying PC-3 ank of sildenafil and DOX combination in DU145 (Fig. S3A) and PC- tumors with DOX (1.5 mg/kg, i.p.) reduced tumor volume (Fig. 3 cells (Fig. S3B). 6A). Sildenafil (5 mg/kg, i.p.) cotreatment potentiated DOX- induced tumor volume reduction (Fig. 6A). The ratio of tumor Sildenafil and DOX Enhance Intrinsic Pathway of Apoptosis. DOX weight to body weight was also reduced with sildenafil cotreatment increased caspase-3 activity that was further enhanced by (Fig. 6B). Similar results were obtained when sildenafil (10 mg/kg)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1006965107 Das et al. Downloaded by guest on September 26, 2021 The synergy of killing of PC-3 and DU145 by sildenafil and DOX A A 25 DOX Sildenafil Fraction CI Cell (µM) (µM) Affected 20 *

1.0 5 0.25 0.45 15 PC-3 2.0 10 0.34 0.72 cells itive 3.0 15 0.47 0.83 10 (% cells) total of DCF pos 0.5 5 0.23 0.46 5 DU145 1.0 10 0.31 0.74 1.5 15 0.45 0.81 0 r r h -48 hr -72 hr rol-48 hr ld rol-72 h ld nt DOX-48 Si nt DOX-72 hrSi o o C C B DOX+Sild-48 hr DOX+Sild-72 hr

n=3 B 25 *p<0.001 vs control and Sild 20 a 20 p<0.001 vs DOX )% 15 ( 15 hta

e * Dl 10 a 10

l * eC 5 * (%cells) total of 5 DCF positivecells 0 l 0 o X ld ld rtn O iS i D S r r r r r o + hr hr h C X 4 4 h 8h 8h 8 8 O l-2 -24 h -2 -4 -4 D d ld ol d ld-4 tr DOX-24 hrSil n DOX-4 Sil Si ontro X+Si o X+ C O C O D D PHARMACOLOGY Fig. 2. (A) Table showing the synergy of PC-3 and DU145 cell killing by Fig. 3. Sildenafil (Sild) enhances DOX-induced intracellular ROS generation sildenafil and DOX performed by colony formation assays using median dose in prostate cancer cells. (A)HDCFDA-positive PC-3 cells after 48 h and 72 h effect isobologram analysis. (B) Sildenafil (Sild) does not enhance DOX-in- 2 of treatment with DOX (1.5 μM) ± Sild (10 μM) (*P < 0.01 vs. other groups duced cell death in normal PrEC. PrEC cell death assessed by trypan blue α after 48-h treatment and P < 0.001 vs. other groups after 72-h treatment; exclusion assay after 24 h of treatment with DOX (1 μM) with/or without Sild n = 4). (B)HDCFDA-positive DU145 cells after 24 h and 48 h of treatment (10 μM) (*P < 0.001 vs. control and Sild and αP < 0.001 vs. DOX; n =3). 2 with DOX (0.2 μM) ± Sild (10 μM) (*P < 0.01 vs. other groups after 24-h α treatment and P < 0.001 vs. other groups after 48-h treatment; n = 4).

was administered daily by oral gavage and DOX (3 mg/kg, i.p.) was A B injected twice per week for 3 wk. (Fig. S5 and ). to trauma sustained by the cavernosal nerve (29). PDE-5 inhibitors DOX treatment increased apoptosis in tumors that was further have been shown to improve erectile function in men postradical amplified by cotreatment with sildenafil (Fig. 6C and Fig. S6A). prostatectomy (30–32). We have established a powerful car- Sildenafil alone had no effect on apoptosis in tumors. Immuno- dioprotective effect of PDE-5 inhibitors in animal models (33). histochemistry demonstrated that the active form of caspase 3 was Moreover, sildenafil improved DOX-induced left ventricular (LV) induced in tumors from sildenafil- and DOX-treated mice com- dysfunction and cardiomyocyte apoptosis (11). In the present study, pared with DOX-treated or nontreated control mice (Fig. 7A). we provide evidence that sildenafil potentiates DOX-induced kill- These data further support in vitro results that show a similar A ing of androgen-independent human prostate cancer cells in vitro trend of increased expression of activated caspase 3 (Fig. 5 )in fi prostate cancer cells. and in vivo. Moreover, sildena l attenuated DOX-induced cardiac dysfunction in mice bearing prostate tumors. These results suggest fi Sildenafil Ameliorates DOX-Induced Cardiac Dysfunction. Cardiac that sildena l may represent a therapeutic approach to improve fi function in mice was assessed by performing echocardiography. A DOX ef cacy in prostate cancer while simultaneously reducing fi slight increase in left ventricular end diastolic diameter (LVEDD) the risk of cardiomyopathy. Our data also show that the sildena l and left ventricular end systolic diameter (LVESD) were observed and DOX combination enhanced the killing of ovarian cancer and with DOX (Fig. S6 B and C). Fractional shortening (LVFS) and sarcoma cells, suggesting a potential utility of sildenafil in chemo- ejection fraction (LVEF) declined in DOX-treated mice (Fig. 7 B sensitization in multiple malignancies. and C). Sildenafil cotreatment with DOX improved LVFS and Mitochondrial ROS is the key component of antitumor activity LVEF compared with the DOX-treated group (P < 0.05) (Fig. 7 B of DOX in tumor cells (25, 26). In the present study, we observed and C). No differences in heart rate were observed between higher levels of intracellular ROS in PC-3 and DU145 cells after control, DOX, and DOX and sildenafil groups (Fig. S6D). Silde- treatment with DOX and sildenafil compared with DOX alone. In nafil-treated animals showed lower heart rates compared with contrast, however, sildenafil and DOX treatment decreased ROS other groups (P < 0.01; n =8)(Fig. S6D). These data suggest that production in normal cells. Similar to these results, sulindac, changes in LVFS or LVEF were independent of heart rate. a potent anticancer drug, selectively enhanced killing of cancer cells exposed to oxidizing agents via production of ROS (34). It has Discussion been suggested that the basic difference in mitochondrial respi- The high incidence of recurrence and metastasis, as well as the re- ration between normal and cancer cells makes cancer cells more fractory nature of the malignancy to , make hormone sensitive to oxidative stress (35, 36). Exactly how sildenafil sensi- refractory prostate cancer one of the most challenging malignancies tizes cancer cells to amplify DOX-mediated ROS generation is not for therapeutic drug combination studies (28). Surgical resection of clear but needs to be investigated. Interestingly, low levels of the prostate also causes significant risk of erectile dysfunction due sulindac also induced delayed preconditioning response against

Das et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 PC-3 A A 0.5 * B DU 145 l d d * l l l i o i r o 0.4 t X d S r S l t X n i + + O n d O l o S X i X D o C D S O 0.3 O C * D D n=4, *p<0.01 vs Control Bcl-2 0.2 * p<0.001 vs DOX Bcl-xL 0.1

Caspase 3,7 ActivityCaspase 3,7 pBad 11 0.0 * Bad l d d l X d d OX D Sil DO Sil + Sil + Sil 10 Contro X Contro X O Bax DO D 9 PC-3 DU145 Actin 8 7 C 0.9 D * L 0.8 x V l- 6 0.7 M c * B -C 0.6 d d- A A 5 0.5 Bcl-xL 0.4 50 4 0.3 * Actin 0.2 40 CaspaseActivity 9

(%cells) total of 3 0.1 DCF positive cells 0.0 30 2 ol X d d r O D Sil X+Sil 20 Cont O 1 D

Cell Death (%) Death Cell 10 0 0 9 r p V V L L x xL h E as M MV l-xL l- V C C -C 4 hr 4 hr M X-CM Bcl-x Bcl- dn ld 2 2 C - Sild-CMVSi l- d- d DO ld--Bc d- 50 A A X+ DOX- Sild--BcSi ild-24 hr il * Procaspase9 DO DOX-24 S DOX+ +S 40 Actin Contro DOX 30

20

Cell Death (%) Death Cell 10

0 B V V p9 p9 p9 p9 M M MV s s DOX C a as Control Cas Ca C Sild-C dnC dn -dn DOX-CMV d-dn X- il S DOX+Sild-C DO DOX+Sild

Fig. 5. Sildenafil (Sild) enhances DOX-induced activation of caspase 3/7 and caspase 9. Cells were treated with DOX (1.5 μM for PC-3 and 0.5 μMfor DU145) ± 10 μM Sild for 72 h. (A) Caspase 3/7 activity in PC-3 and DU145 cells (*P < 0.001 vs. control and αP < 0.001 vs. DOX; n = 6). (B) Representative immunoblots for Bcl-2, Bcl-xL, Bax, pBad, Bad, and Actin from PC-3 and DU145 cell lysates after 48 h of treatment with DOX and/or Sild. (C) Capase-9 α activity in DU145 cells (*P < 0.001 vs. control and P < 0.001 vs. DOX; n = 4). (D) DU145 cells were infected with Adeno-Bcl-xL and Adeno-empty vector (CMV) for 24 h. (Inset) Bcl-xL overexpression. Bar diagram shows cell death after 24 h of treatment with 0.5 μM DOX ± 10 μM Sild (*P < 0.05 vs. DOX-CMV; n = 4). (E) DU145 cells were infected with Adeno-dnCaspase9 and Adeno-empty vector (CMV) for 24 h. (Inset) Procaspase-9 overexpression. Bar chart represents cell death following overexpression of dnCaspase 9 or empty vector 24 h after treatment with 0.5 μM DOX ± 10 μM Sild (*P < 0.05 vs. DOX-CMV; n =4).

Sild DOX+Sild which may enhance Bad heterodimerization with Bcl-xL thereby promoting DOX-induced apoptosis. The ectopic overexpression of Fig. 4. (A and B) Sildenafil (Sild) reduces DOX-induced intracellular ROS Bcl-xL in DU145 cells suppressed the lethality of sildenafilwith generation in PrEC cells. H2DCFDA-positive cells after 24 h of treatment with α DOX compared with DOX alone, suggesting that down-regulation DOX (1 μM) ± Sild (10 μM) in PrEC cells (*P < 0.01 vs. other groups and P < of Bcl-xL played a significant role in the synergistic interactions 0.001 vs. DOX; n =4). between these therapeutic agents. Sildenafil- and DOX-induced cell killing was also associated with increased caspase-3 and cas- ischemia/reperfusion injury in the heart through up-regulation of pase-9 activity. Overexpression of dominant negative procaspase 9 in DU145 cells blocked the enhanced cell killing by combined putative effectors of cardioprotection including iNOS and HSP27 treatment with sildenafil and DOX compared with DOX alone. (37). In this respect, it appears that sildenafil is very similar to Our results show that sildenafil and DOX treatment also caused sulindac in enhancing the antitumor effect while providing car- significant inhibition of tumor growth and enhanced caspase-3 ac- dioprotection at the same time. tivity as well as apoptosis. The sildenafil and DOX combination also Because resistance to apoptosis is one of the hallmarks of cancer, ameliorated DOX-induced cardiac dysfunction, which is consistent we further investigated the mechanisms of cell death induced by with our previous study showing improved LV function with silde- fi sildena l and DOX. Apoptosis is regulated at points within the nafil in DOX-treated mice (11). In these studies, sildenafilreduced intrinsic pathway by pro- and antiapoptotic proteins, which include cardiomyocyte apoptosis, maintained mitochondrial membrane members of the Bcl-2 family together with mitochondria, cyto- potential, preserved myofibrillar integrity, and prevented electro- chrome c, and caspases (38). The regulation occurs by the balance of cardiogram ST interval prolongation after DOX treatment. intrinsic protein levels and/or their localization within intracellular Similar to other chemotherapeutic agents, the clinical use of compartments (39). In the present study, the increased apoptosis by DOX is hampered by its cardiotoxic effects (40). It impairs the sildenafil and DOX was associated with enhanced expression of clinical response and survival of patients (41). Therefore, rendering proapoptotic proteins Bad and Bax and suppression of Bcl-2 and cancer cells more sensitive to DOX while improving cardiac Bcl-xL. Also, this cotreatment regimen dephosphorylated Bad, function would be an efficient approach to enhance its therapeutic

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1006965107 Das et al. Downloaded by guest on September 26, 2021 A A Control Sild DOX DOX+Sild PC-3 Tumor Volume Control 1400 DOX 1200 Sild )

3 1000 DOX+Sild

800

600

Tumor Growth (mm Growth Tumor 400

200

0 0 2 4 6 8 10 12 14 16 18 20

B C B 70 ** 0.04 40 ** 60 30 50 0.03 * * (%) (%) * 40 20 30 0.02 20 LVEF LVFS 10 10 0.01 0 0

X d l d d rol o il il O Sild tr 0.00 D +Sil DOX S Tumor Weight/Body Weight X on X+S Cont C O DO X ld ld D O i D S Si ontrol + C OX D PHARMACOLOGY Fig. 7. Sildenafil enhances activity of caspase 3 in tumor. (A) Representative images of the immunohistochemical staining for Alexa 488 labeled cleaved caspase 3 in tumors. (Top) Cleaved caspase 3 (green fluorescence), (Middle) C nuclei staining with DAPI, and (Bottom) overlay of both types of staining. Cardiac function was assessed by Doppler echocardiography of mice treated 70 * with Sild (10 mg/kg by oral gavage) everyday and DOX (3 mg/kg i.p.) twice 60 per week. (B) LVFS and (C) LVEF (*P < 0.05 vs. control and Sild and **P < 0.01 ± 50 vs. DOX; n = 8). Results are reported as means SE. 40 30 * Company). Apoptosis in tumor section was analyzed using In Situ Cell Death 20 Detection kit, TMR red (Roche Diagnostics). (% ofCells) Total 10 TUNEL positive cells 0 Measurement of ROS. Following 24 and 48 h of treatment with DOX and/or fi μ Dox Sild sildena l, cells were incubated with 50 MofH2DCFDA (Molecular Probes) in Control Dox+Sild growth medium for 30 min at 37 °C. Cells were rinsed with PBS, and ROS levels were visualized by fluorescence microscope.

Fig. 6. Sildenafil (Sild) potentiates DOX-induced inhibition of prostate tumor Caspase-3 and -9 Activity Assay. Cellviability wasmeasuredusingCellTiter-Fluor xenograft growth. Tumor weight and body weight were measured after 18 d viability assay kit (PromegaCorp.)in a 96-wellplate. Caspase-3/7 and-9 activities of treatment with DOX (1.5 mg/kg, i.p.) and/or Sild (5 mg/kg, i.p.). (A) Tumor were measured in treated cells using Caspase-Glo 3/7 assay and Caspase-Glo-9 < α < growth inhibition (*P 0.01 vs. control and P 0.001 vs. other; n = 8). (B) assay kits (Promega Corp.) according to manufacturer’s protocol. Ratio of tumor weight and body weight (*P < 0.05 vs. control and Sild and αP < 0.001 vs. other; n = 8). Sildenafil enhances DOX-induced apoptosis in In Vivo Tumor Study. TumorsweregeneratedinAthymic male BALB/cAnNCr-nu/ tumors. (C) Bar diagram showing TUNEL-positive cells (*P < 0.001 vs. control α nu mice from National Cancer Institute Developmental Therapeutic Program by and P < 0.001 vs. DOX; n = 3). Results are reported as means ± SE. s.c. injection of PC-3 cells (5 × 106 cells) with 50-μL matrigel matrices (BD Bio- science). Tumors were permitted to grow to a volume of ∼200 mm3 over the effect. Our results suggest a potential utility of sildenafilinen- following 2 wk. The animals received i.p. injections of saline (for control), DOX (1.5 mg/kg) alone, sildenafil (5 mg/kg) alone, or DOX (1.5 mg/kg) and hancing the antitumor efficacy of DOX while attenuating its car- sildenafil (5 mg/kg) everyday, 5 d/wk (Fig. S7). Tumor sizes were measured twice diotoxic effect in prostate cancer. Clinical studies are warranted to weekly. Tumor volume was calculated by ab2/2 where “a” and “b” are the long fully define the importance of combined treatment with DOX and fi and short axes of tumor. The animal protocol was approved by the Institutional sildena l as therapeutic tool in prostate cancer patients. Animal Care and Use Committee of Virginia Commonwealth University.

Materials and Methods Doppler Ecocardiography. Cardiac function in nude mice with tumor xeno- Cell Growth and Death Assay. Cell proliferation and metabolically active PC-3 grafts was monitored by Doppler echocardiography using the Vevo770 im- and DU145 cells were measured by CellTiter 96 AQueous One Solution Cell aging system (VisualSonics) as previously reported (12). Proliferation Assay (Promega Corp.) according to manufacturer’s protocol. The percentage of cell death was measured by trypan blue staining. ACKNOWLEDGMENTS. This work was supported in part by National Institutes of Health Grants HL51045, HL59469, and HL79424 (to R.C.K); Apoptosis Assay. Cell apoptosis was analyzed by TUNEL staining using P01-CA104177, R01-CA108325, and R01-DK52825 (to P.D.); and Mid-Atlantic ApopTag Peroxidase In Situ Apoptosis Detection kit (Chemicon International Affiliate Beginning Grant-in-Aid 0765273U (to A.D.).

Das et al. PNAS Early Edition | 5of6 Downloaded by guest on September 26, 2021 1. Leonetti C, et al. (2007) Therapeutic integration of c-myc and bcl-2 antisense 21. Goluboff ET, et al. (1999) Exisulind (sulindac sulfone) suppresses growth of human molecules with in a preclinical model of hormone-refractory prostate prostate cancer in a nude mouse xenograft model by increasing apoptosis. Urology 53: cancer. Prostate 67:1475–1485. 440–445. 2. Singal PK, Li T, Kumar D, Danelisen I, Iliskovic N (2000) Adriamycin-induced heart 22. Lim JT, et al. (1999) Sulindac derivatives inhibit growth and induce apoptosis in failure: Mechanism and modulation. Mol Cell Biochem 207:77–86. human prostate cancer cell lines. Biochem Pharmacol 58:1097–1107. 3. Rivera E (2003) Liposomal anthracyclines in metastatic : Clinical update. 23. Lim JT, Piazza GA, Pamukcu R, Thompson WJ, Weinstein IB (2003) Exisulind and Oncologist 8(Suppl 2):3–9. related compounds inhibit expression and function of the androgen receptor in 4. Shen F, et al. (2008) Quantitation of doxorubicin uptake, efflux, and modulation of human prostate cancer cells. Clin Cancer Res 9:4972–4982. multidrug resistance (MDR) in MDR human cancer cells. J Pharmacol Exp Ther 324: 24. Narayanan BA, et al. (2007) Exisulind in combination with modulates 95–102. epidermal growth factor receptor, cyclooxygenase-2, and cyclin D1 against prostate 5. Bender AT, Beavo JA (2006) Cyclic nucleotide phosphodiesterases: Molecular carcinogenesis: In vivo evidence. Clin Cancer Res 13:5965–5973. regulation to clinical use. Pharmacol Rev 58:488–520. 25. Mizutani H, Tada-Oikawa S, Hiraku Y, Kojima M, Kawanishi S (2005) Mechanism of 6. Das A, Ockaili R, Salloum F, Kukreja RC (2004) Protein kinase C plays an essential role apoptosis induced by doxorubicin through the generation of hydrogen peroxide. Life in sildenafil-induced cardioprotection in rabbits. Am J Physiol Heart Circ Physiol 286: Sci 76:1439–1453. H1455–H1460. 26. Tsang WP, Chau SP, Kong SK, Fung KP, Kwok TT (2003) Reactive oxygen species 7. Das A, Xi L, Kukreja RC (2005) Phosphodiesterase-5 inhibitor sildenafil preconditions mediate doxorubicin induced -independent apoptosis. Life Sci 73:2047–2058. adult cardiac myocytes against necrosis and apoptosis. Essential role of nitric oxide 27. Wang HG, et al. (1999) Ca2+-induced apoptosis through calcineurin dephosphorylation signaling. J Biol Chem 280:12944–12955. of BAD. Science 284:339–343. 8. Ockaili R, Salloum F, Hawkins J, Kukreja RC (2002) Sildenafil (Viagra) induces powerful 28. Pinto AC, Moreira JN, Simões S (2009) Ciprofloxacin sensitizes hormone-refractory cardioprotective effect via opening of mitochondrial K(ATP) channels in rabbits. Am J prostate cancer cell lines to doxorubicin and docetaxel treatment on a schedule- – Physiol Heart Circ Physiol 283:H1263 H1269. dependent manner. Cancer Chemother Pharmacol 64:445–454. fi 9. Salloum F, Yin C, Xi L, Kukreja RC (2003) Sildena l induces delayed preconditioning 29. Rambhatla A, Kovanecz I, Ferrini M, Gonzalez-Cadavid NF, Rajfer J (2008) Rationale through inducible nitric oxide synthase-dependent pathway in mouse heart. Circ Res for phosphodiesterase 5 inhibitor use post-radical prostatectomy: Experimental and – 92:595 597. clinical review. Int J Impot Res 20:30–34. fi 10. Salloum FN, Ockaili RA, Wittkamp M, Marwaha VR, Kukreja RC (2006) Vardena l: A 30. Mydlo JH, Viterbo R, Crispen P (2005) Use of combined intracorporal injection and novel type 5 phosphodiesterase inhibitor reduces myocardial infarct size following a phosphodiesterase-5 inhibitor therapy for men with a suboptimal response to ischemia/reperfusion injury via opening of mitochondrial K(ATP) channels in rabbits. J sildenafil and/or vardenafil monotherapy after radical retropubic prostatectomy. BJU Mol Cell Cardiol 40:405–411. Int 95:843–846. 11. Fisher PW, Salloum F, Das A, Hyder H, Kukreja RC (2005) Phosphodiesterase-5 inhibition 31. Ohebshalom M, Parker M, Guhring P, Mulhall JP (2005) The efficacy of sildenafil with sildenafil attenuates cardiomyocyte apoptosis and left ventricular dysfunction in citrate following radiation therapy for prostate cancer: Temporal considerations. a chronic model of doxorubicin cardiotoxicity. Circulation 111:1601–1610. J Urol 174:258–262, discussion 262. 12. Salloum FN, et al. (2008) Sildenafil (Viagra) attenuates ischemic cardiomyopathy and 32. Teloken PE, Ohebshalom M, Mohideen N, Mulhall JP (2007) Analysis of the impact of improves left ventricular function in mice. Am J Physiol Heart Circ Physiol 294: androgen deprivation therapy on sildenafil citrate response following radiation H1398–H1406. therapy for prostate cancer. J Urol 178:2521–2525. 13. Das A, Xi L, Kukreja RC (2008) Protein kinase G-dependent cardioprotective 33. Kukreja RC, et al. (2005) Pharmacological preconditioning with sildenafil: Basic mechanism of phosphodiesterase-5 inhibition involves phosphorylation of ERK and mechanisms and clinical implications. Vascul Pharmacol 42:219–232. GSK3beta. J Biol Chem 283:29572–29585. 14. Das A, Salloum FN, Xi L, Rao YJ, Kukreja RC (2009) ERK phosphorylation mediates 34. Resnick L, Rabinovitz H, Binninger D, Marchetti M, Weissbach H (2009) Topical sulindac sildenafil-induced myocardial protection against ischemia-reperfusion injury in mice. combined with hydrogen peroxide in the treatment of actinic keratoses. JDrugs – Am J Physiol Heart Circ Physiol 296:H1236–H1243. Dermatol 8:29 32. 15. Piazza GA, et al. (2001) Exisulind, a novel proapoptotic drug, inhibits rat urinary 35. Deberardinis RJ, Sayed N, Ditsworth D, Thompson CB (2008) Brick by brick: – bladder tumorigenesis. Cancer Res 61:3961–3968. Metabolism and tumor cell growth. Curr Opin Genet Dev 18:54 61. 16. Pusztai L, et al. (2003) Phase I and II study of exisulind in combination with 36. Vander Heiden MG, Cantley LC, Thompson CB (2009) Understanding the Warburg – in patients with metastatic breast cancer. J Clin Oncol 21:3454–3461. effect: The metabolic requirements of cell proliferation. Science 324:1029 1033. 17. Whitehead CM, et al. (2003) Exisulind-induced apoptosis in a non-small cell lung 37. Moench I, Prentice H, Rickaway Z, Weissbach H (2009) Sulindac confers high level cancer orthotopic lung tumor model augments docetaxel treatment and contributes ischemic protection to the heart through late preconditioning mechanisms. Proc Natl to increased survival. Mol Cancer Ther 2:479–488. Acad Sci USA 106:19611–19616. 18. Sarfati M, et al. (2003) Sildenafil and vardenafil, types 5 and 6 phosphodiesterase 38. Kroemer G, Reed JC (2000) Mitochondrial control of cell death. Nat Med 6:513–519. inhibitors, induce caspase-dependent apoptosis of B-chronic lymphocytic leukemia 39. Kelly MM, Hoel BD, Voelkel-Johnson C (2002) Doxorubicin pretreatment sensitizes cells. Blood 101:265–269. prostate cancer cell lines to TRAIL induced apoptosis which correlates with the loss of 19. Zhu B, Vemavarapu L, Thompson WJ, Strada SJ (2005) Suppression of cyclic GMP- c-FLIP expression. Cancer Biol Ther 1:520–527. specific phosphodiesterase 5 promotes apoptosis and inhibits growth in HT29 cells. 40. Bast A, Kaiserová H, den Hartog GJ, Haenen GR, van der Vijgh WJ (2007) Protectors J Cell Biochem 94:336–350. against doxorubicin-induced cardiotoxicity: Flavonoids. Cell Biol Toxicol 23:39–47. 20. Li H, et al. (2002) Pro-apoptotic actions of exisulind and CP461 in SW480 colon tumor cells 41. Bryant J, et al. (2007) Use of cardiac markers to assess the toxic effects of anthracyclines involve beta-catenin and cyclin D1 down-regulation. Biochem Pharmacol 64:1325–1336. given to children with cancer: A systematic review. Eur J Cancer 43:1959–1966.

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