Blebbistatin Modulates Prostatic Cell Growth and Contractility through Myosin II Signaling
Ping Chen1, De-qiang Xu1, Sheng-li Xu1, He Xiao1, Sheng-hong Wan2, Xing-huan Wang1, Michael E DiSanto3 and Xin-hua Zhang1*.
1 Department of Urology, Zhongnan Hospital of Wuhan University; Wuhan, China.
2 Department of Urology, First Hospital of Xiaogan, Xiaogan, China.
3 Department of Surgery and Biomedical Sciences of Cooper Medical School of Rowan University, Camden, NJ, USA.
# Ping Chen, De-qiang Xu and Sheng-li Xu contributed equally to this work. *Corresponding Author and person to whom reprint requests should be addressed:
Dr. Xin-hua Zhang M.D., Ph.D.
Department of Urology, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan 430071, P.R. China; Tel: 0086 27 67813043; Email: [email protected]
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ACCEPTED MANUSCRIPT
Abstract
Objectives. To investigate the effect of blebbistatin (BLEB, a selective myosin inhibitor) on regulating contractility and growth of prostate cells and to provide insight into possible mechanisms associated with these actions.
Methods. BLEB was incubated with cell lines of BPH-1 and WPMY-1 and was intra-prostatically injected into rats. Cell growth was determined by flow cytometry and in vitro organ bath studies were performed to explore muscle contractility. Smooth muscle (SM) myosin isoform (SM1/2, SM-A/B and LC17a/b) expression was determined via competitive RT-PCR. SM myosin heavy chain (MHC), non-muscle (NM) MHC isoforms (NMMHC-A and NMMHC-B) and proteins related to cell apoptosis were further analyzed via Western blotting. Masson’s trichrome staining was applied to tissue sections.
Results. BLEB could dose-dependently trigger apoptosis and retard the growth of BPH-1 and WPMY-1. Consistent with in vitro effect, administration of BLEB to the prostate could decrease rat prostatic epithelial and SM cells via increased apoptosis. Western-blotting confirmed the effects of BLEB on inducing apoptosis through a mechanism involving MLC20 dephosphorylation with downregulation of Bcl-2 and upregulation of BAX and cleaved caspase 3. Meanwhile, NMMHC-A and NMMHC-B, the downstream proteins of MLC20, were found significantly attenuated in BPH-1 and WPMY-1 cells, as well as rat prostate tissues. Additionally, BLEB decreased SM cell number and SM MHC expression, along with attenuated phenylephrine-induced contraction and altered prostate SMM isoform composition with upregulation of SM-B and downregulation of LC17a, favoring a faster contraction.
Conclusion. Our novel data demonstrates BLEB regulated myosin expression and functional activity. The mechanism involved MLC20 dephosphorylation and altered SMM isoform composition.
Keywords: Blebbistatin, apoptosis, myosin, prostate, MLC20
Introduction
Benign prostatic hyperplasia (BPH) is a common pathologic process with prevalence increasing with age. Histologic evidence shows that approximately 50% to 60% of men in their 60s, and 80% to 90% in their 70s and 80s (1) have an increased number of epithelial and stromal cells in the periurethral area of the prostate. Thus BPH is correctly referred to as hyperplasia and not hypertrophy. The observed increase in cell number may be due to an imbalance between prostatic cell proliferation and programmed cell death leading to cellular accumulation and prostatic hyperplasia. Although the etiology of BPH is not well understood, androgen levels and aging are associated with the development of BPH (2).
BPH consists of two physiological components: static (increased prostate size) (3) and dynamic (increased prostatic smooth muscle (SM) tone) (4). In the past decades, medical therapies extensively investigated for BPH and lower urinary tract symptoms (LUTS) included α-adrenergic blockers, 5α-reductase inhibitors, aromatase inhibitors, phosphodiesterase inhibitors (PDEIs) and numerous plant extracts. Although, the first line therapies, including α-adrenergic blockers (decreasing prostatic SM tone) and 5α-reductase inhibitors (reducing prostate volume), and their use in combination, are effective and widely prescribed for the treatment of BPH/LUTS, side effects are frequently reported, including dizziness, asthenia and sexual dysfunction. Moreover, approximately 30% of BPH patients need surgical treatments (1). There is thus a need to identify new therapeutic targets for the treatment of BPH/LUTS.
Myosins constitute a superfamily of motor proteins that play an important role in several cellular processes that require force and translocation (5). Recent analyses of genomic databases have shown that most myosins in eukaryotic cells belong to class II (6, 7). Muscle cells mainly contain cardiac, skeletal and SM myosin IIs confined to individual muscle cells. Our previous studies demonstrated that SM cells exhibit a scattered distribution in rat prostatic stroma and α1-adrenergic agonists induce a contractile response (8). SM myosin II (SMM II) is composed of a pair of myosin heavy chains (MHC) and two pairs of myosin light chains (MLC17 and MLC20) that are intimately intertwined (9). Both the 3’ and 5’ end of the MHC pre-mRNA are alternatively spliced to generate COOH-terminal isoforms (SM1 and SM2) and NH2-terminal isoforms (SM-A and SM-B), respectively (10, 11). MLC17 can also be alternatively spliced and has two 3’ end isoforms (LC17a and LC17b) (12, 13). The SMM isoform composition has been demonstrated to affect force development (14) as well as force maintenance (15). The SM-B, LC17a and SM2 isoforms are associated with a faster more phasic-type contraction (e.g., urinary bladder), whereas the SM-A, LC17b and SM1 isoforms are associated with a slower more tonic force generation (e.g., aorta) (16-20). Our recent study found that normal rat prostate contained almost similar SM-B (58.8%), more LC17a (83.8%) and less SM2 (11.4%) compared to their alternatively spliced counterparts, favoring
an intermediate tonicity profile. In addition, myosin II molecules that resemble their muscle counterparts are also present in all non-muscle eukaryotic cells (21-23). Previous studies also determined that non-muscle (NM) myosin II (NMM II) plays a central role in regulating cell migration, cytokinesis and tissue architecture because of its position downstream of convergent signaling pathways (24-27). It is abundantly present in the normal rat prostate (28) and its expression was greatly increased (by 4.5-fold) in the bladder of a rat model of partial bladder outlet obstruction (29). Thus, NMM II might contribute to the pathophysiological processes of some diseases. BPH has been suggested to be a “reawakening” of embryonic processes in which the stromal cell’s inductive potential dictates differentiation of new epithelial gland formation, which is normally seen only in fetal development (30-34). Because there is higher expression of NMM II in embryonic or newborn tissues compared to adult tissues, and the observation that the relative percentage of the NMM II is decreased significantly as the animals reached maturity, NMM II has been referred to as embryonic myosin (35-38). NMM II molecules are comprised of three pairs of peptides: two MHCs, two regulatory light chains that regulate NMM II activity and two essential light chains that stabilize the heavy chain structure. The NM MHC isoforms in mammalian cells result from three different genes (MYH9, MYH10 and MYH14) encoding NM MHC proteins (NMMHC-A, NMMHC-B and NMMHC-C, respectively) (39-42). Although these myosin isoforms are referred to as ‘non-muscle’ myosin IIs to distinguish them from their muscle counterparts, they are also present in muscle cells, where they have distinct functions during skeletal muscle development and differentiation (43), as well as in the maintenance of SM tone (44, 45). The actin-myosin II interaction in SM and NM cells is regulated by the phosphorylation of serine 19 of the 20-kDa light chain of myosin II (MLC20) (46-48) through the activity of actin-activated, Mg2+–dependent ATPase activity of myosin II (46). A number of studies have shown that MLC20 phosphorylation/dephosphorylation plays a central role in cell motility (49-52), endothelial (53, 54) and epithelial (55-57) barrier function, cell division (58-60) and cell apoptosis (61).
Blebbistatin (BLEB), an ATPase inhibiting agent discovered by means of a high throughput small molecule screen for inhibitors of NMMHC-A (62), was recently reported to be a cell permeable selective in vitro myosin II inhibitor in striated muscle and non-muscle cells (IC50 = 0.5 – 5 μM) but reported to be a poor inhibitor of purified turkey gizzard SMM II (IC50, ~ 80 μM) (63). The fact that the four amino acid residues identified as the BLEB binding site on NMMHC-A and SMM II were found to be identical (64) suggested that there should be potent inhibition of SMM II by BLEB. Indeed, BLEB has been suggested to inhibit SM contraction with near equipotency as for NMM II (15, 28, 65, 66). In addition, Straight et al. demonstrated that BLEB blocked cell blebbing and inhibited contraction of the cleavage furrow without disrupting mitosis or contractile ring assembly (62). Meanwhile, at
concentrations of 25 and 50 μM, Fazal et al. found that BLEB dose-dependently induced apoptosis by MLC20 dephosphorylation through inhibition of myosin ATPase activity in porcine pulmonary artery SM cells (61). However, several studies indicate a role for BLEB in protection of damaged mammalian cells. Croft et al. found that 50 μM BLEB inhibited TNFα/CHX induced apoptotic nuclear breakdown and membrane blebbing in fibroblasts (67) and Wang et al. found that myosin IIA-actin complex contributes to membrane blebbing during H2O2-induced neuronal apoptosis and 1 μM BLEB could restore H2O2-induced apoptosis (68). The different effects of BLEB on apoptosis might be attributed to the myosin IIA-actin complex, which is involved in a positive feedback loop that links caspase-3/ROCK1/MLC signaling axis (68).
To date, no studies have directly addressed the regulation of BLEB on cell proliferation and apoptosis in the prostate, as well as prostatic SM contractile characteristics. The study presented here aimed to elucidate the effect of BLEB on prostatic myosin II signaling and its effect on cell growth and SM contractility.
Materials and Methods
Animals and tissues: A total of thirty-six specific-pathogen-free (SPF) grade adult male Sprague-Dawley rats weighing 300–350 g were used. All surgical procedures were performed under anesthesia by intra-peritoneal injection of pentobarbital sodium (35 mg/kg; Abbott Laboratory, Chicago, IL). A stock solution of BLEB (Ellisville, MO, USA) was made in dimethyl sulfoxide (DMSO). Rats underwent small midline incisions of the lower abdomen above the penis and the ventral prostates were exposed. With a 30-gauge needle, three increasing doses (0.05, 0.1 and 0.2 nmoles) of BLEB in a final volume of 50 μl sterile normal saline were injected into both right and left ventral lobes of the prostate. For the control rats, 50 μl sterile normal saline with nearly commensurable DMSO was injected. Accordingly, the rats were divided into four groups (0, 0.05, 0.1 and 0.2 nmoles BLEB-treated groups) (n = 9 in each group). After the injection, a 2% lidocaine solution was applied to the wound, and then the wound was closed. Two weeks post-surgery rats were euthanized; ventral prostates, bladders and seminal vesicles were harvested and weighed. Prostatic strips of approximately 1 cm × 1 cm × 0.5 cm were prepared (the surrounding prostatic capsule along with excess fatty tissue was dissected free) for organ bath physiology studies and immediately placed in Krebs-Henseleit (Krebs) solution and the remaining tissue frozen in liquid nitrogen or saved at −80°C for subsequent molecular analyses or put into 10% neutral buffered formalin for histological examination. All animal protocols were approved by the Animal Experiment Center of Zhongnan Hospital of Wuhan University.
Human prostatic cell lines: SV40 large-T antigen-immortalized stromal cell line WPMY-1 (Cat. #GNHu36) was purchased from the Stem Cell Bank, Chinese Academy of
Sciences in Shanghai, China. Human benign prostatic enlargement epithelial cell line BPH-1 (Cat. #BNCC339850) was purchased from the Procell Co., Ltd. in Wuhan, China. Verfification of the cell lines was performed at the China Centre for Type Culture Collection in Wuhan, China. The BPH-1 cells were cultured in RPMI-1640 medium (Gibco, China) containing 10% fetal bovine serum (FBS) (Gibco, Australia), WPMY-1 cells were cultured in DMEM medium (Gibco, China) containing 1% penicillin G sodium/streptomycin sulfate and 5% FBS in a humidified atmosphere consisting of 95% air and 5% CO2 at 37ºC. After culture for 24 h, BPH-1 and WPMY-1 cells were treated with the myosin II selective inhibitor BLEB (0, 5 and 10 μM) for 48 h. In addition, BPH-1 and WPMY-1 cells were pretreated for 1 h with
50 μM z-VAD-fluoromethyl ketone (z-VAD-fmk), a cell-permeable caspase inhibitor (61, 69). These cells were subsequently treated with 5 μM BLEB for 48 h.
Determination of cell viability: Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, 100 µl cell suspensions (2 × 104 cells per ml) with 10% FBS medium was seeded to a 96-well plate
and incubated for 3 days at 37ºC. Then 20 μl MTT was added (5 mg/mL final concentration in medium) and the cells were incubated for another 4 h at 37 °C. The medium was removed and 150 μl DMSO was added to each well which was then shaken for 10 min at room temperature to completely dissolve the blue-purple precipitate. The absorbance was measured at 490 nm using a Microplate Reader (Thermo, USA).
Flow cytometry analysis for alterations of cell cycle and apoptosis: For cell cycle analysis, 1 × 106 cells were harvested and fixed in 70% ice cold ethanol at -20°C for overnight. After centrifugation, pellets were resuspended with PBS containing 50 μg/ml propidium iodide and 0.1 mg/ml RNaseA in the dark. After incubation at 37°C for 30 min, the DNA content distribution was analyzed by flow cytometry analysis (Beckman, Cat. #FC500). For cell apoptosis analysis, 1 × 106 cells were harvested and then analyzed by flow cytometry analysis using FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA), according to the manufacturer’s instructions.
In vitro organ bath studies: Rat prostatic strips were mounted longitudinally in 20 ml ZW-SX Digital thermostats smooth groove (Wuxi Woshin instruments, Jiangsu, China). Strips were equilibrated at least 1 h in Krebs buffer at 37ºC with continuous bubbling with 95% O2 and 5% CO2. The buffer had the following mM composition: NaCl 110, KCl 4.8, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25 and dextrose 11 and was changed every 15 min. Strips were continuously adjusted to 300-600 mg resting tension (66). After equilibration, prostate tissue was contracted with 60 mM KCl, washed, and then contracted with increasing concentrations (10-7-10-4 M) of phenylephrine (PE). Force produced by the above stimuli was normalized to tension (% KCl).
Total RNA extraction and cDNA synthesis: As previously described (66), total RNA was isolated from the frozen tissues using TRIzol reagent according to the manufacturer’s protocol. The resulting RNA was quantitated by spectrophotometry at 260/280 nm using a NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA, USA). 1 µg of RNA was converted to complementary DNA (cDNA) using reverse transcriptase via the SuperScript II First-Strand Synthesis System according to the manufacturer (Invitrogen, Waltham, Massachusetts, USA).
Competitive reverse transcriptase polymerase chain reaction (competitive
RT-PCR): SM-A/SM-B, SM1/SM2 and LC17a/LC17b alternatively splice isoforms were amplified with competitive PCR, using a T100™ Thermal Cycler (Bio-Rad, Hercules, CA, USA). The primer sequences are shown in Table 1. The cycling conditions were an initial 5 min at 94°C followed by 35 cycles (30 s at 94°C, 30 s at 55°C, and 60 s at 72°C), ended by a final one-time 7 min incubation at 72°C to ensure extension of all products. The PCR products were then separated by electrophoresis on a 2.5% agarose gel and were visualized using GelStar staining and ultraviolet illumination. Band density was quantified by Quantity One® SW 1-D Analysis software (Bio-Rad) which enabled us to obtain quantitative relative SM myosin isoform expression data for all isoform pairs.
SDS-PAGE and Western Blotting Analysis: As previously described (70), proteins were extracted from frozen cells and tissues using Radio-Immunoprecipitation Assay lysis buffer (Sigma-Aldrich, St Louis, Mo) with freshly added phenyl methane sulfonyl fluoride (PMSF) and sodium orthovanadate. 100 μg of each sample was eletrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel and transferred to polyvinylidene fluoride membrane (PVDF) (Millipore, Billerica, MA, USA) using a Bio-Rad wet transfer system. The membrane was blocked for 2 h at room temperature with Tris-buffered saline with 0.1% [v/v] Tween containing 5% [w/v] non-fat dry milk solution. The membrane was incubated overnight with the corresponding protein primary antibodies (Table 2). After washing, the membranes were incubated with secondary antibody at room temperature for 2 h. Detection of reaction antigen was performed with an enhanced chemiluminescence (ECL) kit (Thermo Fisher Scientific, Waltham, MA, USA). The bands were quantified by Quantity One® SW 1-D Analysis software (Bio-Rad).
Masson’s trichrome staining: As previously described (71), rat prostate tissues fixed in 10% [v/v] neutral buffered formalin for 24–36 h were processed for paraffin embedding. The paraffin-embedded tissue sections (5 µm) were processed for histologic observation by Masson’s trichrome staining. Prostatic SM cells, collagen fibers and epithelial cells were stained dark red, blue and red, respectively. In each sample, we analyzed three areas under magnification (×100).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay: Rat prostate tissues were fixed in 4 % paraformaldehyde, embedded in paraffin, and then digested with proteinase K for 20 min. The sections were then incubated with a fluorometric DNA fragmentation detection kit (PromoCell, Heidelberg, Germany) according to the manufacturer’s instructions. Nuclei were labeled with DAPI (4′, 6-diamidino-2-phenylindole). DAPI and fluorescence-labeled images were merged and TUNEL-positive apoptotic cells in the merged images were quantified by the counting of positively stained cells.
High Performance Liquid Chromatography (HPLC)
A quantity of 0.2 nmoles BLEB was injected into both left and right ventral lobes of the prostate and then the serum BLEB concentrations of rats were measured after being treated at 0, 0.5, 1, 3, 7, 14 post-injection days using HPLC assay as previously described with minor modification (72). Briefly, serum samples were extracted into chloroform/methanol and then vacuum dried. The extract was then dissolved with 70% methanol. Chromatographic detection of the compound was performed using an Agilent 1260 Infinity II (Agilent Technologies, Santa Clara, CA, USA) liquid chromatography system. The chromatogram acquisition and integration of the compound data was processed by ChemStation software.
Statistical analysis: Results are expressed as mean ± SEM. Statistical analysis used either the Student’s t-test with Excel software (two sample treatments compared) or ANOVA and Bonferroni post-tests with GraphPad Prism 5.0 (multiple means compared). P < 0.05 was considered to be significant.
Results
BLEB inhibited cell growth of prostatic cells via inducing apoptosis
As BLEB has a cytotoxic effect, we detected survival rates of BPH-1 and WPMY-1 cells after BLEB treatment and calculated the half maximal inhibitory concentration (IC50) of BLEB for the cytotoxic effect using an MTT assay (Supplemental figure 1). We found the IC50 for the cytotoxic effect of BPH-1 and WPMY-1 cells is 36.5 μM and 38.2 μM, respectively. Therefore, we chose 5 μM and 10 μM dosages for our experiments utilizing human prostate cell lines and 0.05, 0.1 and 0.2 nmoles BLEB for in vivo intra-prostatic injection.
The MTT assay revealed that BLEB dose-dependently inhibited the cell growth of both BPH-1 (Figure 1a) and WPMY-1 (Figure 1f) with a significant difference observed at 48 h. To better understand the underlying mechanisms, the effect of BLEB on cell apoptosis and cell cycle was determined using flow cytometry analysis. Both dosages of BLEB treatment resulted in a significant increase in the number of apoptotic human epithelial and stromal cell lines (Figures 1b–1d and 1g–1i). BLEB resulted in similar rates of apoptosis in both cell lines. BLEB did not trigger cell cycle arrest in either cell line (Figure 2). We further analyzed the alterations of proteins involved in the apoptosis using Western-blotting (Figure 3) and found
the upregulation of the apoptosis inducer BAX and downregulation of the apoptosis inhibitor Bcl-2 in the BLEB-treated BPH-1 and WPMY-1 cells. Meanwhile, caspase 3, a downstream protein of Bcl-2 and BAX in the apoptotic cascade [47], was decreased, with its active form cleaved caspase 3 increased.
The in vivo effect of BLEB treatment was investigated in rat ventral prostates. With detected DAD wavelength being 420 nm, we found the HPLC retention time for 5 μM and 0.25 μM BLEB standard buffers were 14.802 and 14.851 mins, respectively (Supplemental figure 2). After calculation through quantitative analysis, the detectable and credible minimum concentration was considered as 0.125 μM while the serum BLEB concentrations of rats at T0, T0.5, T1, T3, T7 and T14 days were not detected. Thus, BLEB might be retained locally. As shown in Figure 5 and Table 3, after BLEB treatment for 2 weeks, there was no significant difference in body weight, seminal vesicle and bladder weights among the four groups. However, compared to control (BLEB 0 nmoles) group, the ventral prostate weights of animals treated with 0.1 and 0.2 nmoles BLEB were significantly decreased (by 38.7% (P < 0.05) and 50.7% (P < 0.01), respectively). Also, differential histopathology was observed among four dosage groups with Masson’s trichrome staining. In the BLEB-treated groups, the atrophy of the prostate was mainly due to loss of prostatic epithelial and SM cells with a relative increase of collagen fibers) (Figures 6). Interestingly, loss of prostatic epithelial cells occurred in 0.1 and 0.2 nmoles BLEB-treated groups, while the loss of SM cells was observed only in 0.2 nmoles BLEB-treated group. Thus, rat prostatic epithelial cells might be more sensitive to BLEB than SM cells in vivo. Additionally, a TUNEL assay showed apoptosis rates were significantly and dose-dependently increased in the three BLEB-treated groups compared to control (BLEB 0 nmoles) group (Figure 7). Similar to human prostatic cell lines, after BLEB treatment, BAX and cleaved caspase 3 had significantly increased expression, while Bcl-2 and caspase 3 expression was decreased in a dose-dependent manner (Figure 9c).
BLEB induced downregulation of non-myosin II via inhibiting MLC20 phosphorylation
The isoforms of non-myosin II, NMMHC-A and NMMHC-B, were determined after BLEB treatment with Western blotting (Figure 3 and Figure 9c). NMMHC-A and NMMHC-B were found significantly attenuated in BPH-1 and WPMY-1 cells, as well as rat prostate tissues. Moreover, p-MLC20, the active upstream protein of NMMHC-A and NMMHC-B, exhibited decreased expression yet with relatively over-expression of the nonphosphorylated MLC20.
BLEB induced apoptosis activated by cleaved caspase 3 through MLC20 dephosphorylation
The BPH-1 and WPMY-1 cells were pre-treated with 50 μM z-VAD-fmk in order to repress the activity of cleaved caspase 3 followed by treatment of the cell lines with 5 μM BLEB. Flow cytometry demonstrated that this treatment resulted in a significant lowering of cell apoptosis in both BPH-1 and WPMY-1 cells (Figures 4a and 4b). Western blotting further demonstrated that the 5 μM BLEB increased level of cleaved caspase 3 was attenuated by 50 μM z-VAD-fmk but that z-VAD-fmk did not block the BLEB decrease of p-MLC20 expression (Figure 4c). These observations suggest that MLC20 could be an upstream protein of caspase 3 and that MLC20 dephosphorylation could induce prostatic cell apoptosis through activation of cleaved caspase 3.
BLEB decreased prostatic SM contractile response
In the current study, animals treated with 0.2 nmoles of BLEB were found to have significantly less SM MHC expression (Figure 9c) similar to the Masson staining showing less SM cells in the 0.2 nmoles BLEB-treated group (Figure 6). PE-mediated contraction was also investigated. Prostatic SM per se generated significant force in response to KCl depolarization and PE-mediated adrenergic stimulation in a dose-dependent manner (Figure 8). BLEB treatment attenuated PE-induced contraction but it was observed only in the 0.2 nmoles dosage group, which was consistent with the decreased expression of SM MHC observed by Western-blotting.
BLEB altered prostatic SM contractility by regulating SMM isoforms
Interestingly, the time required to reach 50% PE-mediated maximum contraction of rat prostates in the 0, 0.05, 0.1 and 0.2 nmoles BLEB-treated groups were 13.5 ± 1.4 S, 11.2 ± 1.0 S, 10.7 ± 0.8 S and 10.5 ± 0.9 S, respectively (Table 4), suggesting a faster contraction in the BLEB-treated prostates. Thus, the isoforms of SMM were further analyzed using competitive RT-PCR. As shown in Figures 9a and 9b, the relative expression of SM-B to SM-A was increased and LC17a to LC17b was decreased, with no observed change in SM1/2 expression in BLEB-treated prostates, correlating with a faster contractile velocity.
Discussion
Our studies are the first to report that BLEB modulates myosin II expression and functional activity in the prostate. We provide evidence suggesting that BLEB can induce prostatic apoptosis via activation of cleaved caspase 3 through MLC20 dephosphorylation with NMMHC-A and NMMHC-B being less expressed and subsequently inhibition of cell growth in vitro and in vivo. BLEB also attenuated PE-mediated prostatic SM contractility and altered prostate SMM II isoform composition with upregulation of SM-B but downregulation of LC17a, favoring a faster contraction.
BLEB, a selective myosin ATPase inhibiting agent (62), was originally assumed to be more effective on the activity of NMM II, which plays a key role in the control of cell
migration, cytokinesis and tissue architecture (24-27). In this study, we found BLEB treatment significantly attenuated the growth of both BPH-1 and WPMY-1 cells in vitro and caused rat prostate atrophy in vivo. The BLEB inhibitory effect is similar between cultured epithelial cells and SM cells, but in vivo study showed the epithelial cells appear to be more sensitive to BLEB than the SM cells. This discrepancy could be attributed to different species; i.e. cultured human cells are different from rat tissue. Interestingly, previous studies have also found that the apoptosis of rat epithelial and SM cells were not synchronous after castration and rat SM cells were less sensitive to castration (8, 73, 74). Therefore, rat SM cells might be more resistant to cell apoptosis induced by BLEB.
Flow cytometry analysis further demonstrated that BLEB induced growth inhibition was caused by apoptosis and not associated with cell cycle arrest. As a crucial mediator of cell cycle arrest and cell apoptosis (75-78), p53 contributes to tumor progression modulated by NMMHC-A (79). Additionally, MLC20 dephosphorylation was assumed to involve a BLEB-NMM mediated apoptosis process. Fazal and colleagues found that inhibiting myosin ATPase activity with BLEB resulted in MLC20 dephosphorylation and induced cell apoptosis in porcine pulmonary artery SM cells(61). In our current study, we demonstrated that p-MLC20, the active upstream protein of NMM, levels were lower with a relatively elevated MLC20 expression. Moreover, when the activity of cleaved caspase 3 was repressed with z-VAD-fmk, BLEB treatment did not induce apoptosis. Accordingly, Western blotting demonstrated that there was a reduction in cleaved caspase 3 with no any alteration of p-MLC20. Therefore, BLEB-NMM triggered apoptosis could be attributed to MLC20 dephosphorylation. In general, myosin II ATPase activity is distal to the cell death pathway and diminishing MLC20 phosphorylation decreases ATP hydrolysis and subsequently induces apoptosis (47, 48).
BLEB was also found to attenuate both the expression of NMMHC-A and NMMHC-B. As an upstream protein of NMM, attenuated p-MLC20 could decrease NMM expression. There are three NMM isoforms, namely NMMHC-A, NMMHC-B and NMMHC-C. Besides a role of NMMHC-A in modulating P53 stability, NMMHC-A also behaves as a normal, but slow, conventional myosin with a low duty cycle, spending most of its kinetic cycle detached from actin (80). Unlike NMMHC-A, NMMHC-B may be better adapted for maintaining tension in a static manner (81, 82). NMMHC-C is relatively recently identified NMM isoform that has relatively low expression in SM organs when compared to nervous tissue (42) and therefore was not investigated in the present study.
As BLEB has a cytotoxic effect, we calculated the IC50 of BLEB for the cytotoxic effect and found the IC50 for the cytotoxic effect of BPH-1 and WPMY-1 cells is 36.5 μM and 38.2 μM, respectively. Therefore, in this current study, we chose 5 μM and 10 μM dosages for human prostate cell lines and 0.05, 0.1 and 0.2 nmoles quantities of BLEB for in vivo
intra-prostatic injection. Thus, the increased apoptosis in the prostate and the decreased levels of myosins' expressions and MLCs' phosphorylations are directly affected by BLEB's myosin inhibitory effect, instead of a cytotoxic effect.
Two studies have described a role of BLEB in damaged mammalian cells. Croft et al. found that 50 μM BLEB inhibited TNFα/CHX induced apoptotic nuclear breakdown and membrane blebbing in fibroblasts (67). Also, Wang et al. showed that the myosin IIA-actin complex contributes to membrane blebbing during H2O2-induced neuronal apoptosis and 1 μM BLEB could attenuate H2O2-induced apoptosis (68). The different effect of BLEB on apoptosis might be attributed to the myosin IIA-actin complex, which is involved in a positive feedback loop that links to the caspase-3/ROCK1/MLC signaling axis (68).
Although BLEB was originally reported to be a selective inhibitor of the myosin II isoforms expressed by striated muscles and non-muscle (IC50 = 0.5–5 μM) but a poor inhibitor of purified turkey smooth muscle myosin II (IC50, ~ 80 μM) (63), our previous studies have clearly demonstrated that BLEB is indeed also a potent inhibitor for rat corpora cavernosum, bladder and prostate SM in vitro (15, 28, 66). Consistent with the decreased number of SM cells and the expression of SM MHC, in vivo BLEB treatment with the high dose (0.2 nmols) significantly decreased the response of isolated prostatic strips to PE stimulation.
In addition to the altered force generation, isolated prostatic strips from BLEB-injected groups produced a faster contraction. Accordingly, the relative expression of SM-B, suggested to be an important role in increasing velocity of cell shortening (14, 83), was found to be strongly increased after BLEB treatment in the current study. Meanwhile, LC17a was found at reduced levels following BLEB treatment. Although several studies have concluded that relative higher ratios of the LC17a to LC17b isoform correlate with faster phasic contraction (17, 84, 85), Rovner et al. (86) reported an approximately two-fold higher in vitro motility speed and Mg2+-ATPase activity in expressed SM-B heavy meromyosin regardless of the LC17 isoforms present (pure LC17a or pure LC17b). Sweeney et al. (87) reported that in vitro motility sliding velocity and actin-ATPase activity correlate with increasing loop size/flexibility but were not affected by the LC17 isoforms present. Interestingly, X-ray crystallography data have revealed that the LC17a/b can approach the 25/50-kDa loop, which is the location of the NH2-terminal SM-A/-B isoforms and near the nucleotide binding pocket of myosin and regulates unloaded shortening velocity in SM tissues (84, 87). Thus, shortening velocities may not only be determined by the LC17 isoforms but could also be affected by differences in SM-A/-B isoform (88).
A limitation for the current study is that the protein levels of the SMM II isoforms were not determined because at present SM-A/SM-B antibodies are not commercially available. However, a previous study demonstrated that mRNA levels of SMM isoforms correlated well
with protein expression (89). Another limitation is that pharmacokinetic properties of BLEB are not available, and it is quite difficult to directly detect the BLEB concentrations of rat prostate tissues using an HPLC assay. Indeed, Young et al. reported that a single intra-BLC (basolateral amygdala complex) infusion of the NM II inhibitor BLEB produced a long-lasting disruption of context-induced drug seeking for at least 30 days in rats (90, 91). Thus, a single intra-prostatic injection of BLEB might produce a long-lasting myosin II inhibiting effect. In addition, as BLEB has a cytotoxic effect and is unstable, along with insoluble in aqueous conditions, the effect on prostate of the novel blebbistatin derivatives, para-aminoblebbistatin, para-nitroblebbistain and azidoblebbistatin, is of great interest for future investigation.
In conclusion, our molecular, physiological and histological data demonstrate that BLEB regulates SMM II and NMM II expression and functional activities of rat prostate. BLEB modulated NM MHC isoforms expression with attenuation of NMMHC-A and NMMHC-B, which could then affect cell apoptosis via activation of cleaved caspase 3 through MLC20 dephosphorylation. Similar to NMM II, BLEB decreased SM MHC expression and PE-mediated contraction. BLEB also altered prostate SMM isoform composition with upregulation of SM-B and downregulation of LC17a, favoring a faster contraction. Our study indicated that BLEB not only decreased prostate size (static component) but also changed the prostatic SM tone (dynamic component). These effects suggest that BLEB might be developed as a therapeutic agent for treating BPH.
Author Contributions: Xin-hua Zhang, Michael E DiSanto, Xing-huan Wang and Ping Chen participated in research design; Ping Chen, De-qiang Xu and Sheng-hong Wan conducted experiments; Ping Chen, Sheng-li Xu and He Xiao performed data analysis; Xin-hua Zhang, Michael E DiSanto and Ping Chen wrote or contributed to the writing of the manuscript.
This research is supported by National Natural Science Foundation of China (N.
81770757 and N. 81270843).
Disclosures Statement: The authors have nothing to disclose.
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Figures and Legends
Figure 1. Decreased growth of BPH-1 and WPMY-1 cells treated with BLEB via increased apoptosis. Panels a and f shows cell viability of BPH-1 and WPMY-1 cells treated with 0 (black, solid circle), 5 (red, solid square) and 10 (blue, solid triangle) μM BLEB at different time points of 0, 24, 48 and 72 h by MTT assay. For 5 μM BLEB vs. 0 μM BLEB, *
= 0.01 < p < 0.05, ** = p < 0.01. For 10 μM BLEB vs. 0 μM BLEB, ## = p < 0.01. Panels b, c and d are the flow cytometry analyses for apoptosis of BPH-1 cells treated with selected concentrations of 0, 5 and 10 μM BLEB for 48 h, respectively. Panel e is the bar graph of the apoptosis rate (%) of BPH-1 cells based on panels b, c and d. ** = p < 0.01. Panels g, h and i
are the flow cytometry analyses for apoptosis of WPMY-1 cells treated with selected concentrations of 0, 5 and 10 μM BLEB for 48h, respectively. Panel j is the bar graph of the apoptosis rate (%) of WPMY-1 cells based on panels g, h and i. ** = p < 0.01.
Figure 2. Flow cytometry analysis for cell cycle in BPH-1 and WPMY-1 cells treated with BLEB. Panels a, b, and c are the flow cytometry analyses for cell cycle in BPH-1 cells treated with selected concentration of 0, 5 and 10 μM BLEB for 48 h, respectively. Panel d is the bar graph for the percentage of BPH-1 cells in each phase. NS means no significant difference between groups. Panels e, f, and g are the flow cytometry analyses for cell cycle in WPMY-1 cells treated with selected concentration of 0, 5 and 10 μM BLEB for 48 h, respectively. Panel h is the bar graph for the percentage of WPMY-1 cells in each phase. NS means no significant difference between groups.
Figure 3. Expression of representative proteins altered in BPH-1 and WPMY-1 cells after BLEB treatment. Western blotting analyses of proteins (NMMHC-A, NMMHC-B, MLC20, p-MLC20, caspase 3, cleaved caspase 3, Bcl-2 and BAX) altered in BPH-1 and WPMY-1 cells after 48 h treatment of BLEB. GAPDH was used as a loading
control.
Figure 4. Recovery of BLEB-induced cell apoptosis in BPH-1 and WPMY-1 cells pre-treated with 50 μM z-VAD-fmk. Panels a and b are the bar graphs for BLEB-induced apoptosis rate (%) of BPH-1 and WPMY-1 cells, respectively. Cells are treated with 0 μM BLEB (white bar), 5 μM BLEB (grey bar) and 5 μM BLEB + 50 μM z-VAD-fmk (black bar) for 48 h. ** = p < 0.01 and NS means no significant difference between groups. Panel c shows expression of proteins (MLC20, p-MLC20, caspase 3 and cleaved caspase 3) altered in BPH-1 and WPMY-1 cells pre-treated with 50 μM z-VAD-fmk. GAPDH was used as a loading control.
Figure 5. Typical photographs of rat urogenital tissues. Panels a, b, c and d are the rat urogenital tissues from control (BLEB 0 nmoles, top left) and BLEB-treated rats with concentration of 0.05 nmoles (top right), 0.1 nmoles (bottom left) and 0.2 nmoles (bottom right), respectively. (1) ventral prostate, (2) seminal vesicle, (3) bladder.
Figure 6. Masson’s trichrome staining of prostate tissue. Prostatic SM cells were stained dark red, collagen fibers were stained blue and epithelial cells were stained red. Panels a, b, c and d are the Masson’s trichrome staining for the prostates from control (BLEB 0 nmoles) and 0.05, 0.1 and 0.2 nmoles BLEB-treated rats, respectively (magnification × 200). Panel e is the bar graph for area percentage of different tissue components (SM, epithelia and collagen fibers) (n = 6 different animals for each group). * = 0.01 < p < 0.05 vs. control group (BLEB 0 nmoles), ** = p < 0.01 vs. control group (BLEB 0 nmoles). NS means no significant difference between groups.
Figure 7. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay detecting cell apoptosis of prostate tissues. Panels a, b, c and d shows the TUNEL staining for the prostates from control (BLEB 0 nmoles) and 0.05, 0.1 and 0.2 nmoles BLEB-treated rats, respectively (magnification × 100). Positive staining area and cell numbers are quantified by Image J software. Panel e is the bar graph for apoptosis rate (%) of TUNEL positive cells in rat prostate (n = 6 different animals for each group). ** = p < 0.01 vs. control (BLEB 0 nmoles).
Figure 8. Contractility of rat ventral prostate. The summary graph of phenylephrine (PE) induced prostatic SM contraction normalized by KCl induced contraction (n = strips obtained from different animals in each group, one strip was used for each animal). * = 0.01 < p < 0.05, ** = p < 0.01 and NS means no significant difference between groups. The x-axis represents concentration (M) of PE while the y-axis represents tension (%KCl).
Figure 9. The expression of SM myosin II isoforms and representative proteins in rat prostate. Panel a shows the competitive RT-PCR bands of smooth muscle myosin II (SMM II) isoforms (SM-A/-B, SM1/2, LC17a/b) in rat ventral prostate in each group. Panel b shows the bar graph for the expression of SM-A/-B, SM1/2 and LC17a/b in each group (n = 6 different animals for each group). ** = p < 0.01 vs. control (BLEB 0 nmoles) and NS means no significant difference between groups. Panel c is the Western blotting analyses of proteins (SM MHC, NMMHC-A, NMMHC-B, MLC20, p-MLC20, caspase 3, cleaved caspase 3, BAX and Bcl-2) altered in rat prostates after 2-week BLEB treatment. GAPDH was used as a loading control.
Supplemental figure 1: Typical dose-response curves for the inhibition of
proliferation by BLEB in BPH-1 (a) and WPMY-1 cells (b). The x-axis represents
concentration (μM) of BLEB while the y-axis represents cell inhibition (%).
Supplemental figure 2: BLEB concentrations were detected by high performance
liquid chromatography (HPLC). Standard BLEB retention time for 5 μM and 0.25 μM were
14.802 (a) and 14.851 mins (b), respectively. c shows the standard curve for BLEB
determined by HPLC.
Table 1. Primer sequences used to amplify target genes by PCR.
Target gene Primer Primer sequence
SM-A/-B forward 5′-GGCCTCTTCTGCGTGGTGGTC -3′
reverse 5′-TTTGCCGAATCGTGAGGAGTTGTC -3′
LC17a/b forward 5′-GAGAGTGGCCAAGAACAA-3′
reverse 5′-CAGCCATTCAGCACCATGCG-3′
SM1/2 forward 5′-GCTGGAAGAGGCCGAGGAGGAATC-3′
reverse 5′-GAACCATCTGTGTTTTCAATAA-3′
Abbreviations: SM, smooth muscle; LC, light chain.
Table 2. List of primary antibodies.
Antigens Species antibodies Dilution Supplier
raised in (WB)
SM MHC mouse monoclonal 1:1000 Santa Cruz, sc-6956
NMMHC-A rabbit polyclonal 1:1000 Abcam, ab75590
NMMHC-B rabbit monoclonal 1:1000 Abcam, ab204358
NMMHC-C rabbit monoclonal 1:1000 Abclonal, A3690
MLC20 rabbit polyclonal 1:1000 Cell Signaling Technology, #3672
p-MLC20 rabbit polyclonal 1:1000 Cell Signaling Technology, #3671
caspase 3 rabbit monoclonal 1:1000 Cell Signaling Technology, #9662
cleaved caspase 3 rabbit monoclonal 1:1000 Cell Signaling Technology, #9664
BAX rabbit monoclonal 1:1000 Cell Signaling Technology, #5023
Bcl-2 rabbit monoclonal 1:1000 Cell Signaling Technology, #2872
β-actin mouse monoclonal 1:1000 Santa Cruz, sc-47778
Table 3. Variation of physiological parameters in BLEB-treated (0.05, 0.1, 0.2 nmoles) and control (0 nmoles) rats.
Body weight (g) Ventral Seminal Bladder weight
Group prostate vesicles
Initial Final (mg)
weight (mg) weight (mg)
BLEB 0 nmoles 302.5±33.3 404.1±29.9 672.5±124.6 445.0±25.0 130.0±12.3
BLEB 0.05 nmoles 311.0±8.5 384.4±20.4 476.0±69.3 445.0±35.7 142.5±4.3
BLEB 0.1 nmoles 310.5±33.6 382.1±20.1 412.5±108.7* 425.0±26.0 132.5±8.3
BLEB 0.2 nmoles 303.8±17.9 384.9±29.0 331.5±96.6** 427.5±14.8 122.5±10.9
Abbreviations: BLEB, blebbinstatin. P values calculated by unpaired t test. Data are mean ± SD. * P < 0.05 vs. sham rats, ** P < 0.01 vs. sham rats.
Table 4. Time to 50% phenylephrine (PE) mediated maximum contraction for prostate in 0, 0.05, 0.1 and 0.2 nmoles BLEB-treated groups.
Group Number Time (s) P value (compared to BLEB 0 nmoles)
BLEB 0 nmoles 8 13.5 ± 1.4 NA
BLEB 0.05 nmoles 7 11.2 ± 1.0 < 0.01
BLEB 0.1 nmoles 7 10.7 ± 0.8 < 0.01
BLEB 0.2 nmoles 8 10.5 ± 0.9 < 0.01
Abbreviations: NS, not applicable; PE, phenylephrine. Results for time to 50% phenylephrine (PE) mediated maximum contraction are expressed as mean ± SEM.