Ultrasound Targeted Microbubble Destruction for Novel Dual Targeting of HSP 72 and HSC 70 in Prostate Cancer

Prostate cancer is the most frequent malignancy and a leading cause of cancer-related deaths in American men, accounting for an estimated 238, 590 new cases and 29,720 deaths in 2013 (Siegel et al., 2013; DeSantis et al., 2013). It also is the second leading cause of cancer deaths among men, with an estimated 903, 000 newly diagnosed cases and 258,000 deaths per year worldwide. Recently, the incidence of PCa increases steadily in China (Jemal et al., 2011). Androgen deprivation therapy (ADT) provides an effective therapy for patients with advanced PCa. Although most patients with advanced PCa show an initial response to ADT, a significant percentage of patients invariably progress to hormone refractory prostate cancer (HRPC) and castration-resistant prostate cancer (CRPC) (Bitting et al., 2013; Garcia et al., 2012). Responses to systemic chemotherapy are frequently poor (Di Lorenzo et al., 2007). Therefore, additional therapies are needed. Another challenge is that PCa are also associated with poor prognosis and androgen independence. Effective targeting


Introduction
Prostate cancer is the most frequent malignancy and a leading cause of cancer-related deaths in American men, accounting for an estimated 238, 590 new cases and 29,720 deaths in 2013 (Siegel et al., 2013;DeSantis et al., 2013).It also is the second leading cause of cancer deaths among men, with an estimated 903, 000 newly diagnosed cases and 258,000 deaths per year worldwide.Recently, the incidence of PCa increases steadily in China (Jemal et al., 2011).Androgen deprivation therapy (ADT) provides an effective therapy for patients with advanced PCa.Although most patients with advanced PCa show an initial response to ADT, a significant percentage of patients invariably progress to hormone refractory prostate cancer (HRPC) and castration-resistant prostate cancer (CRPC) (Bitting et al., 2013;Garcia et al., 2012).Responses to systemic chemotherapy are frequently poor (Di Lorenzo et al., 2007).
Therefore, additional therapies are needed.Another challenge is that PCa are also associated with poor prognosis and androgen independence.Effective targeting

Targeting of HSP72 and HSC70 in Prostate Cancer
Hang-Hui Wang & , Yi-Xin Song & , Min Bai, Li-Fang Jin, Ji-Ying Gu, Yi-Jin Su, Long Liu, Chao Jia, Lian-Fang Du* treatment for advanced CRPC and HRPC remains a critical clinical challenge.Thus, there is a clear need for novel targeted therapeutic strategies for the treatment of advanced PCa. siRNA have provided us with dawn to tackle this issue.The properties of siRNA that are attractive for therapeutics include (i) stringent targetgene specificity, (ii) relatively low immunogenicity of siRNA and (iii) simplicity of design and testing of siRNA (McNamara JO 2nd et al., 2006).
The HSP70 family includes at least eight members with diverse biochemical functions, including nascent protein folding, preventing denatured protein aggregation, and modulating assembly/disassembly of protein complexes (Daugaard et al., 2007;Zuiderweg et al., 2013).The exact role of the HSP70 family in cancer remains to be elucidated.Firstly, The two major cytoplasmic isoforms are HSC70 and HSP72.Generally, HSC70 is abundantly and ubiquitously expressed in nontumor tissues, whereas HSP72 is present at relatively low levels in the absence of stress.Secondly, HSC70 and HSP72 expression may reduce sensitivity to HSP90 inhibitors.Combinatorial modulation of these two HSP70 isoforms could therefore be doubly advantageous (Davenport et al ., 2010;Powers et al., 2010;Meng et al., 2011;Stangl et al., 2011;Evans et al., 2010;Balaburski et al., 2013;Rérole et al., 2011).
However, the specific and efficient delivery of siRNA into cancer cells in vivo remains a major obstacle (Yao et al., 2012).Therefore, development of a useful multi-modal approach or multiple pathways in targeted delivery will further improve siRNA efficacy.
UTMD has been recognized as an efficient modality for drug and gene delivery in vive and in vitro.Microbubble destruction by ultrasound exposure generates microstreams or microjets that create shear stress on cells and open transient pores in cell membranes (Li et al., 2009;Tachibana et al., 1999;Xie et al 2010;Suzuki et al., 2011;Zheng et al., 2012).
In the present study, we tested whether UTMD combined with dual targeting of HSC70 and HSP72 could promote the specific and efficient delivery of siRNA, and to explore its molecular mechanism.

Cell and Cell culture
The human prostate carcinoma cell line VCaP and prostate epithelial cell RWPE-1 cell line were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured accordingly.VCaP cell lines were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (GIBCO), 100 U/ml penicillin and 100 U/ml streptomycin (Invitrogen) and maintained in a humidified atmosphere of 5% CO 2 , 95% air at 37 °C.RWPE-1 cells were grown in Keratinocyte serum-free medium (K-SFM) medium (Invitrogen) supplemented with 5 ng/ml human recombinant EGF and 0.05 mg/ ml bovine pituitary extract under standard cell culture conditions.

UTMD exposure protocols
A therapeutic ultrasound machine (PHYSIOSON-Basic, PHYSIOSON Elektro-medizin, Germany) was used to emit ultrasound at the frequency of 1 MHz.The area of the ultrasound probe was 2.5 cm 2 .The ultrasound transducer was placed at the bottom of plates or dishes with coupling medium on the surface of the transducer.The adjustable sonication parameters included ultrasound intensity, exposure time, pulse frequency, and duty cycle.Microbubbles (SonoVue, Bracco, Milan, Italy) were lipid-shelled ultrasound contrast agents containing sulfur hexafluoride gas (diameter 2.5 -6.0 µm) and used at a concentration of ~2×10 8 bubbles/mL.The volumetric ratio of microbubbles to medium dictated the choice of contrast agent dose.

siRNA-mediated gene knockdown
siRNA (Shanghai GenePharma Co., Ltd) were designed against the open reading frame of HSP72 (HSPA1A; accession number NM_005345) or HSC70 (HSPA8; accession number NM_006597).Two active sequences were used for studies against HSP72 or HSC70 (designated HSP72 or HSC70).Active sequences for HSP72 and HSC70 are as follows: HSP72 (sense:GGACGAGUUUGAGCACAAGTT, antisense: CUUGUGCUCAAACUCGUCCTT), HSC70 (sense:CCAAGCAGACGCAGAUCUUTT, antisense: AAGAUCUGCGUCUGCUUGGTT), Negative control (sense:UUCUCCGAACGUGUCACGUTT, antisense: ACGUG ACACGUUCGGAGAATT).For all experiments cells were transfected with siRNA (200 nM for single transfections or 100 nM for combinatorial transfections) in Opti-MEM® medium without serum according to the Lipofectamine® 2000 protocol (Invitrogen).An equal amount of scrambled Stealth siRNA was used as a negative control.Six hours after the cells were transfected, the medium was replaced with fresh culture medium.All experiments were performed 48 h after transfection and repeated three times.

Gene transfection efficiency
Following gene transfection, the effciency of transfection was evaluated by fuorescence microscopy and flow cytometry.Green fuorescence was detected using inverted fuorescence microscopy (Zeiss Axiovert S100; Carl Zeiss, Jena, Germany).The percentage of FAM expression of the transducted VCaP cells was quantitatively measured by fow cytometry (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA)

Cell viability assays
A cell counting kit-8 assay (CCK-8, Dojindo Laboratories) was used to evaluate relative cell viability.At 48 hours after siRNA transfections, RWPE-1 and VCaP cells were plated in 96-well microtiter plates at a density of 5×103 cells per well, then the live cell count was assayed using CCK-8 according to the manufacturer's instructions.Briefly, 10 µL of CCK8 solution was added to each well, and the absorbance at 450 nm was measured using a Thermomax microplate reader (Molecular Devices, Hercules, CA) after 1-4 hour incubation.Relative cell viability was calculated as a percentage of untreated control cells.

Flow Cytometry
Cells were trypsinized and resuspended in Opti-MEM medium and plated on 6-well plates at 1, 000 cells/well after transfection with siRNA for 48 h.For apoptosis detection, cells were stained with Annexin V and propidium iodide using the Annexin V-FITC Apoptosis Detection kit (Invitrogen), and the percentage of apoptotic cells was determined by flow cytometry (Beckman Coulter).

Statistical analysis
Statistical analysis was carried out with SPSS version 12.0 (SPSS, Inc., Chicago, IL, USA).All data were represented as mean ± SD and analyzed by One-way ANOVA.Values were considered statistically significant at P<0.05.Results were representative of more than three individual experiments.

HSP72, HSC70, and HSP90 is overexpressed in human prostate cancer cells compared with human prostate epithelial cells
We compared gene expression profiles of VCaP and RWPE-1in vitro, and found that HSP72, HSC70, and HSP90 was overexpressed in VCaP cells.Western blot analysis revealed that VCaP cells expressed higher levels of HSP72, HSC70, and HSP90 protein in vitro compared with RWPE-1, which expressed undetectable levels.VCaP and RWPE-1cells expressed very low levels of cleaved caspase-3 (Figure 1).

Effects of HSP70 expression after transfection at different time points
To confirm the optimal time points in HSP70 expression in vitro after transfection, we selected 24 h, 48 h, 72 h, 96 h to quantifiy the expression of HSP70 protein in VCaP cells.As shown in Figure 2, optimal time points of efficacy of silencing HSP70 gene by siRNA is 48 h.Therefore, we set up 48 h to further investigate after transfection in vitro.

UTMD is a safety and noninvasive approach
In order to investigate the safety of UTMD, cellular viability was monitored using the cell counting kit-8 assay for 48 hours.The cellular viability of VCaP cells transfected with HSP72 and HSC70 siRNA was delayed compared with that of the control groups after UTMD.No significant difference was found in the cellular viability between the RWPE-1 and VCaP of the control group and the two group treated with UTMD (Figure 4B).
To investigate the molecular mechanisms by which the combination of UTMD and HSP70-siRNA restrains PCa in the context of HRPC, we process a set of relevant experiment in vitro system.RT-qPCR revealed 3.1-fold higher levels of HSP72, HSC70, and HSP90 mRNA in NC control compared with HSP72/HSC70-siRNA treatment (Figure 5).The group of UTMD combined HSP72/HSC70-siRNA expressed undetectable levels of HSP70 and HSP90 mRNA.This is consistent with the results of Western Blot in VCaP cells (Figure 6).To better understand the biological processes that underlie what appears to be HSP70, HSP90, and cleaved caspase-3 in VCaP cells, We next sought to define the mechanism by which HSP70 regulates cleaved caspase-3 expression.we used flow cytometry and found that the cleaved caspase-3 gene that was most significantly enriched in the group of UTMD combined HSP72/HSC70-siRNA (Figure 4).Therefore, we tested the ability of the combination of UTMD with HSP72/HSC70-siRNA to induce tumor

Discussion
Silencing of gene expression by siRNA is rapidly becoming a powerful tool against cancer, efficient delivery of siRNA into tumor cells remains a key obstacle (Pai et al., 2006).HSP90 is an exciting therapeutic in cancer because inhibition of this single protein causes the simultaneous degradation of multiple oncoproteins and combinatorial blockade of numerous oncogenic pathways.HSP70 molecular chaperones are of interest when considering modulation of HSP90 (Whitesell et al., 2005).HSP70 is the major therapeutic in advanced PCa (Alaiya et al., 2001;Garrido et al., 2003;Lebret et al., 2003;McConnell et al., 2013;Matthias et al., 2013).However, targeting HSP70 alone can result in off-target effects, drug resistance and disease recurrence.Therefore, simultaneous targeting of a multi-modal approach or multiple pathways in targeted delivery could in principle be an effective approach to treating PCa.With respect to the functional role of HSP70 isoforms in the HSP90 chaperone, The data we presented here demonstrate that silencing HSC70 or HSP72 individually has no effect on HSP90 protein level of VCaP prostate cancer cells, while dual targeting of HSC70 and HSP72 inhibit the cellular chaperone activity of HSP90.Additionally, UTMD combined with simultaneous targeting of HSC70 and HSP72 may further enhance silencing efficacy of the expression of HSP70, HSP90 protein and mRNA.In agreement with our findings, Powers et al. (2008) also found that dual targeting of HSC70 and HSP72 silence HSP90 protein in human HCT116 colon and A2780 ovarian cells.
In the present study, we have shown that silencing HSC70 or HSP72 individually has no effect on apoptosis by flow cytometry.Using combinatorial siRNA approach, we revealed that dual silencing of HSC70 and HSP72 considerably increased the apoptotic efficacy (Figure 4A, 6), UTMD may further induce extensive tumor-specific apoptosis.our apoptosis results were in accord with the study of Rérole et al. (2011).The data of apoptosis is nearly consistent with the cleaved caspase-3 of quantitative Real-Time PCR and western blot.Importantly, the safety and noninvasive of the combinational targeting strengthen our confidence further.And these data indicate that this is a promising strategy for delivering siRNA as cancer therapeutics.
The most important attribute of UTMD combination with siRNA is their specific, efficient delivery, which leads to concentration in the target tumor and avoidance of distribution to irrelevant normal tissues.Specific delivery into the intended target cell will likely reduce the dose required for antitumor activity as well as limit toxicity.
An exciting implication of our research is the attractiveness of combinatorial targeting of HSC70 and HSP72 as an alternative means to achieve HSP90 inhibition, with the added advantage of avoiding the antiapoptotic effects of HSP70 isoform induction that limit the use of current pharmacologic inhibitors.Interestingly, UTMD may promote the effect of silencing HSP90.The approach of UTMD combined siRNA was used here as a tool to silence the expression of HSC70, HSP72, and HSP90.It is possible that UTMD combined with RNA interference may be developed as a potential therapeutic approach.
Our findings also reveals further details about the relationship between UTMD and siRNA, especially in the context of HRPC.The reason of UTMD combined with dual targeting of HSC70 and HSP72 improve the efficacy of silence are as follows: Firstly, UTMD promote the efficient delivery of siRNA.Secondly, UTMD also may activate the active region of HSP70, HSP90 or increase crosstalk between distinct signaling pathways.Further work is required to identify the molecular and cellular mechanisms whereby UTMD interact with siRNA in vitro In summary, this study provide experimental support for the efficacy and therapeutic potential of combination UTMD with dual targeting HSP70-siRNA for the treatment of HRPC in vitro.Because of the specificity and efficacy of we were able to target a gene accurately, without any obvious toxicity.Our work supports the potential role for HSP70 as biomarker targeting in PCa (Powers et al., 2008;Goloudinaet al., 2012).The multi-modal delivery strategy is versatile because the same delivery agent may be better aimed to target cells, an ideal, promising approach for delivering siRNA as cancer targeted therapeutics.
Figure 1.HSP72, HSC70, and HSP90 is Overexpressed in VCaP Compared with RWPE-1.Representative images showing expression of HSP72 HSC70, and HSP90 protein in VCaP and RWPE-1 cell as analyzed by Western blotting (P<0.05).All experiments were repeated three times with similar results

Figure 2 .Figure 3 .
Figure 2. Optimal Time Points of Efficacy of Silencing HSP70 Gene by siRNA-transfected.(A) HSP72 siRNA transfection; (B) HSC70 siRNA transfection.The best time points of efficacy of silencing HSP70 gene was at 48 h ( P<0.05)

Figure 4 .
Figure 4. Apoptosis and Viability Assay of RWPE-1 and VCaP Cells in Presence of UTMD and siRNA.(A)Apoptosis rates of 3 groups shown.Compared with siRNA group and UTMD plus siRNA group (P<0.01).(B) The cellular viability of VCaP cells transfected with HSP72 and HSC70 siRNA was delayed compared with that of the control groups after UTMD (P<0.01).UTMD is a safety and noninvasive approach for RWPE-1