Rosuvastatin inhibit the cell proliferation and the expression of angiogenic factors Vascular Endothelial Growth Factor and Matrix-Metalloproteinase-9 in DU-145 human prostate cancer cells

Background: Pathophysiology angiogenesis refers to uncontrollable endothelium capillary proliferation, it can be seen in diseases such as growth and metastasis of tumors. The aim of this was to study the effects of Rosuvastatin on the cell growth and expression of the Vascular Endothelial Growth Factor (VEGF) and matrix metal proteases (MMP-9) genes in human prostate cancer DU-145 cells. Methods: DU145 cells were cultured in RPMI medium containing 10% FBS, then treated with Rosuvastatin (5-100μM) for 96 hr. The cell proliferation was analyzed by cell counting, MTT and trypan blue tests. Nitric oxide production was assayed by Greis test. The wound healing scratching test was employed to analyze the cell migration. The VEGF and MMP-9 gene expression was analyzed by RT-PCR. Results and conclusion: The Rosuvastatin can cause a higher rate of inhibition in cell proliferation and growth which accomponish by inhibition of cell migration in scratching assay. Considerably, decrease in cell growth and migration was observed in half of the DU145 cells, treated by 25 μM rosuvastatin. NO assay showed that high concentrations of the medication are able to reduce NO production and release. Moreover, VEGF-A and MMP-9 genes expression could be inhibited by 50 μM of Rosuvastatin. Research Article Rosuvastatin inhibit the cell proliferation and the expression of angiogenic factors Vascular Endothelial Growth Factor and Matrix-Metalloproteinase-9 in DU-145 human prostate cancer


Introduction
Prostate cancer can be considered malignant tumor which grows external part of the gland to develop gradually in the internal part, then, is transmitted to all parts of the body via blood fl ow. Although there is no diagnosed symptom in the cancer at the primary stage, the tumor steadily grows to put pressure on urinal, consequently [1]. Tumor tissues are able to adsorb nutrients and oxygen up to one to two millimeters radii, then it is compulsory to form new feeding vessels network [2]. During oncogenesis and cancer progression the cancerous cells can induce to form new vessel from the available arterioles network as similar to normal angiogenesis. It means that more tumor size increases, more the environment becomes hypoxic and oxidized to express and produce increasingly several kinds of growth factors for developing local blood vessels, as the consequence [3]. Angiogenesis can be stimulated in tumor by many involving factors such as growth factors, whether specifi c or non-specifi c for endothelial tissue, could be considered as the angiogenesis inducers in tumor [4].
Vascular endothelial growth factors (VEGFs) plays the main and crucial role to induce angiogenesis. Five members have been reported for VEGFs' family. Some tumor cells release VEGF A protein to affect tumor microenvironment, containing stromal surrounding cells for stimulating VEGF F promoter to express the gene. So, there is a collaboration and correlation between normal and cancerous cells [5,6]. Not only VEGFs can induce the proliferation of the endothelial cells, but also increase their permeability. The aspect can be effective to start angiogenesis, because well-developed and grown vessels have to be unstable, before branching [7].
Also, extracellular matrix metalloproteinases (MMPs) which are playing remarkable roles in the constructive trends of the body structures and forming, effectively involve in tumor angiogenesis and metastasis [8]. Digestion of the basic membrane components such as collagen and fi bronectin, by MMPs, is the most important stage of the polyphase pathway to cause metastasis. Many studies have demonstrated that a high level of MMPs, particularly MMP-2 and MMP-9 which are released by epithelial cells, relates to increased metastasis in prostate cancer [9]. It is considered that MMP-2 and MMP-9 are profoundly expressed by stromal cells, whilst other MMPs are biosynthesized by tumor cells [9]. In contrast, MMPs digest plasminogen to release angiostatin as an inhibitor of angiogenesis. The inhibitor, then, binds to its specifi c receptor on the surface of the endothelial cells. Endostatin, a collagen fragment released by the protolithic activity of the elastase, can also inhibit MMPs [10].

Statins
are the competitive inhibitors of the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase that are widely applied to treat cardiovascular disorders, particularly hyperlipidemia, at the present time. Recent studies demonstrate that statins can signifi cantly act as antioxidant and anti-infl ammatory compounds, independent on the antilipidemic effects [11,12]. Rosuvastatin is a new generation and artifi cial type of the statins. There are many studies to report the anticancer activities of the medications on several types of the cancer cells [13]. Erbaş, et al. evaluated the rosuvastatin effects on the activity of the arginase, polyamines including putrescine, spermidine and spermine, and ornithine levels, in cancer tissue. They found that rosuvastatin can protectively affect breast cancer, as well as inhibition of arginase activity and ornithine and consequently polyamine levels [14]. It is also reported that rosuvastatin can individually inhibit colon adenocarcinomas development. It is also more effective for excluding polyamines and improving the activity of the natural killer cells [15]. El Sayed, et al. could fi nd some signals which are play roles in the antitomur effects of resuvastatin to infl uence positively on hepatocellular carcinoma by inhibiting p-focal adhesion kinase/p-rous sarcoma oncogene cellular homolog cascade [16]. A recent study showed that polymeric nanocapsules loaded with resuvastatin have anticancer activity on hepatocellular carcinoma by enhancing apoptosis [17]. Also, Kheiri, et al. recently used resuvastatin-loaded nanoparticles to suppress MCF-7 cell lines of the breast cancer [18] The literature reviews show that blocking angiogenesis by some small chemical inhibitors might have very limited effect on cancerous cells growth. Interest in fi nding anti-angiogenesis agents for cancer prevention and inhibition is attractive.
Understanding the molecular mechanism and their biological actions are necessary too. Because of some limitation and disadvantage of each agent to inhibition of these processes are under investigation. Therefore, the aim of this work was to investigate the effect of Rosuvastatin on the growth, proliferation and migration of human prostate cancer DU-145 cells and correlation of them by VEGF and MMP-9 genes expression.

Materials and methods
Cell preparation and culture DU-145 cells is a standard cell line of the prostate cancer, used in remedial studies [19]. The cell line was provided from in National Institute for Genetic Engineering and Biotechnology

MTT assay
MTT assay approach was utilized to measure cells proliferation for analysis of the rosuvastatin effects on the cells.
Breifl y, 10μl MTT was added to each well, then the cells were incubated for 4 hours. Then the supernatants were aspirated.
The diformazan dyes which utilized by the cell pelelts were sulibilised by adding 100 μL of DMSO. They were shacked for 15s by the shaker of the ELISA reader and analyzed by the Elisa reader at 580nm.

Measurement of the generation of Nitric Oxide (NO)
The amount of NO generation from DU-145 cells were assayed after treatment by Rosuvastatin. Cell-free supernatants were recovered after incubation. Produced NO was quantitatively measured as NO 3 -(Nitrate: a stable metabolite of NO) and NO 2 -(Nitrite) concentrations by using enzymatic and colorimetric NO assays [19]. In this method, we used reduction of nitrate by commercially Aspergilus niger nitrate reductase and measurement of the product (Nitrite) by Griess reagent in micro titer plate. In brief, 50 μl of cells condition medium or standards were added to each well. Just prior to each assay freshly made solutions of NADPH (

Gene expression
Rosuvastatin treated DU-145 cells were collected and total RNAs were extracted by RNX-Plus Kit (SinaClon Co., Tehran, Iran), according to the manufacture's protocol. Then the quality of the nucleic acids was measured by Nano-drop (Beckman Coulter Inc. CA, USA) at A 260/280 and A230/ 280. After that, RT-PCR, and consequently Real-time PCR, were employed by the designed specifi c primers as described in Table 1 to analyze VEGF and MMP-9 gene. First-strand cDNA was synthesized from 1 μg of total RNA using Maurine Maloney Leukemia virus (M-MLV, Fermentas) and reverse transcriptase (Fermentas Gmbh, Leon-Rot, Germany) with oligo-dT primer (Fermentas Gmbh, Leon-Rot, Germany), according to the manufacturer's instructions.
The expected sizes of the RT-PCR product were 121 bp for GAPDH and 250 bp for VEGF. The thermal cycling conditions for amplifi cation of those fragments were as follows: 94 °C for 10 min., followed by 35 cycles at 94 °C for 50s; 54 °C for 30s; 72 °C for 60s. This was followed by re-extension at 72 °C for 10 min. The PCR products were separated on a 2% agarose gel (using 0.5× TBE buffer) and visualized by ethidium bromide staining. For quantitation each band of gel was scanned by image analysis program (ImageJ. Exe). The expression of the target genes was quantifi ed and then normalized by an endogenous reference housekeeping gene (GAPDH) relative to the calibrator (untreated cells). Relative intensity of each gene mRNA and fold of change referred to untreated control cells.

RT-PCR and real-time quantitative detection of the VEGF and MMP-9 mRNAs
Quantitative real-time PCR was carried out on cDNAs prepared for conventional RT-PCR. We used the ABI PRISMTM 7700 Sequence Detector System (PE Applied Biosystems) and the fl uorescent dye SYBR® Green and the amount of PCR products was determined based on the fl uorescence produced during the extension step of each cycle in a closed tube. To normalize the amount of total RNA present in each reaction, we used GAPDH as housekeeping gene an internal control. The threshold cycle (Ct) value for each sample was proportional to the log of the initial amount of input cDNA. Relative expression levels of VEGF and MMP-9 in each treatment group were derived from normalizing the Ct value of genes against that of an endogenous reference and a calibrator, where by GAPDH was used as a normalizer and untreated DU-145 cells as a calibrator. Quantitative data were analyzed and relative quantifi cation of VEGF and MMP-9 mRNAs were derived by the 2 -ΔΔC T methods, as described by Livak [20].

Quantitative ELIZA for VEGF
The amount of secreted VEGF in response to Rosuvastatin by DU-145 cells was assayed in supernatants by using commercial Calbiochem human VEGF ELISA kits. (Calbiochem-Merck-Bioscience, Darmstadt, Germany) according to the procedure manual of the kits. The method is a ''sandwich'' enzyme immunoassay employing monoclonal and polyclonal antibodies. Quantitation is achieved by construction of a standard curve using known concentrations of VEGF calibrators of the kit.

Scratching assay
Cell migration was measured using the wound healing assay as described in our previous work [21]. Briefl y, equal numbers of DU-145 cells (5×10 5 ) were plated and kept overnight in 6-well plates. After that the monolayer of cells was carefully wounded by manual scratching with a micro-pipette tip head. The cells were treated with different concentration of Rosuvastatin and incubated for 96 hr. The wounded regions were photographed at different times interval (0-96hr) using a phase contrast microscope (Nikon). The photographs of the cells were analyzed using a Cell science software program. The distance between the two sides of scratch layers of images taken from migration of all concentrations at specifi ed times was determined by the Cell science software.

Statistical analysis
The data were statistically analyzed by One-way ANOVA test via Graphed software, and the graphs were drawn by Microsoft Offi ce Excel 2016.

Cell morphology
The response of human prostate cancer cells line DU-145 to Rosuvastatin were initially assayed by cells morphology and behavior, cell growth, viability and MTT assay. The cells morphology was observed by microscope to assay qualitatively the effects of Rosuvastatin on DU145 cells after 96 hr treatment of cells. The phenotypic changes and morphology of the cells in response to Rosuvastatin was shown in Figure 1. With increasing of Rosuvastatin concentration the cell density, confl uency and adherence were decreased. In untreated control cells, the cells fully confl uenced, stretched, spindle-shaped, and without appendages which are pressed together (B). The cells treated by 10,25,50 and 100μM of Rosuvastatin respectively (C,D,E and F). The morphology of treated cells was changed. The cells confl uency were decreased and cell proliferation were suppressed by increase of Rosuvastatin concentration.

Cell proliferation
The cells , viability and growth were determined by using trypan blue exclusion test, cell counting and MTT cell proliferation assays. The result of cell counting show that Rosuvastatin inhibited cell growth potential and cell proliferation parameters up to 10% in the presence of Rosuvastatin 50-100μM in comparison of untreated control cells (R 2 = 0.96) (p<0.05) (Figure 2a). In MTT assay, the absorbance values of 4 independent tests were averaged and growth curves were constructed (Figure 2b). The results indicate that, Rosuvastatin inhibited cell proliferation of DU-145 cells in a linear dose dependent manner (R 2 = 0.97). The cells viability was decreased from 98% to 45% in presence of 5 to 100 μM of Rosuvastatin (Figure 2c). The results indicated that cells were still viable (>70%) after treatment with 25 μM of Rosuvastatin.

Nitric oxide production
The response of DU-145 cells to Rosuvastatin and Nitric Oxide production were assayed in cells supernatant. The supernatants were assessed for yield of released NO. The assays were carried out as explained in methods those factors should tightly controlled to prevent failure of the assay. An assay failure was defi ned as the inability to generate a standard curve in the range of 0.05-2.0 AU. Initially the standard calibration curves of Sodium Nitrite and Sodium Nitrate were plotted. The measured absorbance was plotted against the sodium Nitrate (Figure 3a) or Sodium Nitrite (Figure 3b) concentrations. A higher degree of correlation was observed in standard curves. When the DU-145 cells were treated by Rosuvastatin, the amount of released NO in culture medium was quantifi ed by measurement of NO metabolites (Nitrate and Nitrite) as described in methods. DU-145 cells those treated with Rosuvastatin specially in higher doses, (50-100 μM ), signifi cantly reduced the production and release of NO to 20-30% in comparison to untreated cells.  GTPase signaling [28].

Gene expression and VEGF secretion
Nowadays, there are many increasing studies to report statin effects, as well as cardiovascular treating advantages, such as anti-infl ammation, immune system regulation, and antioxidant activity. Anti-tumor in vitro experiments on model animals have indicated that statins are able to inhibit cancer cell proliferation and induce apoptosis, then reduce tumor growth.
Thus, statins have been considered as a potential medications for cancer treatment, over the last decades. Recent fi ndings show that high concentrations of statins are able to permeate indirectly into the tumor cells to inhibit angiogenesis, however, side effects should be considered [29]. There are some in vitro and in vivo studies to show that fl uvastatin and lovastatin can prevent metastasis and growth of the pancreatic cancer cells, as well as atorvastatin inhibiting metastasis in melanoma cells [30].
As the results of the present study, raising rosuvastatin dose can effectively inhibit the growth of DU145 cells, so that, 100 μM rosuvastatin extremely shows an inhibitory effect on the growth and proliferation of these cells. The medication is able to restrict tumor development vascular because rosuvastatin can reconstruct vessels in systemic malignancies. The drug can also infl uence as an inhibitor on tumor growth and development of malignant prostate cancer. In a recent study, it has been reported that long-term consumption of high doses of rosuvastatin can decrease the risk of breast cancer and, similarly, inhibit breast cancer cell invasion and metastasis [31]. The results of the present study demonstrated that although higher doses of rosuvastatin indicate more inhibitory effects on the expression of the VEFG gene, as an important factor in angiogenesis, there was no inhibitory effect of the drug on the expression of the MMP-9 gene as the other factor contributing in angiogenesis. Many studies have been indicated dual effects of the different doses of the statins on angiogenesis in tumor. These medications, for instance, reduces VEFG production and inhibit the capillary tube formation. As the fi ndings of this study, statins are interestingly able to both enhance and inhibit angiogenesis. In other words, lower concentrations of rosuvastatin enhance angiogenesis, in contrast, inhibitory effects on the angiogenesis could be observed in higher doses of the drug. Therefore, dose plays a key role for enhancing or inhibiting angiogenesis. In another study, it has been reported that there is no same sensitivity to atorvastatin among six cancer cell lines, related to brain, colon, uterus, skin, prostate, and breast.

Conclusions
According to the results, it is concluded that higher concentrations of the Rosuvastatin can cause a higher rate of inhibition in cell proliferation and growth which accomponish by inhibition of cell migration in scratching assay. Considerably, decrease in cell growth was observed in half of the DU145 cells, treated by 25 μM rosuvastatin. NO assay showed that high concentrations of the medication are able to reduce NO production and release. Moreover, VEGF-A and MMP-9 genes expression could be inhibited by 50 μM of Rosuvastatin.

Acknowledgment
This work was supported by research funds (No. 540) from National Institute of Genetic Engineering and Biotechnology, Tehran-Iran.