Octreotide Associated with Apoptotic-Upregulation in
Castration-Resistant Prostate Cancer
Sha Zhu1*, Judith Apondi Oremo1, Sanqiang Li2, Minghui Zhen1, Yue Tang1, Ying Du1
1Department of Microbiology and Immunology, College of Basic Medical Sciences, Zhengzhou University, Zhengzhou, P. R. China,2Department of Biochemistry and Molecular Biology, Medical College, Henan University of Science and Technology, Luoyang, P. R. China
Abstract
Androgen deprivation therapy has become the fist-line treatment of metastatic prostate cancer; however, progression to castrate resistance disease occurs in the majority of patients. Thus, there is an urgent need for improvements in therapy for castration-resistant prostate cancer. The aims of the present study were to determine the efficacy somatostatin analogue octreotide (OCT) combined with a low dose of docetaxel (DTX) using castration resistant prostate cancer cells and to investigate the involved molecular mechanisms in vitro. The anti-proliferative and synergism potential effects were determined by MTT assay. Induction of apoptosis was analyzed employing annexing V and propidium iodide staining and flow cytometry. VEGFA, CASP9, CASP3 and ABCB1 gene expression was evaluated by RT-PCR and Q-RT-PCR analysis. OCT in combination with DTX treatments on DU145 cell migration was also evaluated. Investigation revealed that combined administration of DTX and OCT had significant, synergistically greater cytotoxicity than DTX or OCT treatment alone. The combination of the two drugs caused a more marked increase in apoptosis and resulted in greater suppression of invasive potential than either individual agent. There was obvious increase in caspase 3 expression in the OCT alone and two-drug combined treatment groups, however, VEGFA expression was markedly suppressed in them. These results support the conclusion that somatostatin analogues combined with docetaxel may enhance the chemotherapy efficacies through multiple mechanisms in castration-resistant PCa cell line. This work provides a preclinical rationale for the therapeutic strategies to improve the treatment in castrate resistance disease.
Citation:Zhu S, Oremo JA, Li S, Zhen M, Tang Y, et al. (2014) Synergistic Antitumor Activities of Docetaxel and Octreotide Associated with Apoptotic-Upregulation in Castration-Resistant Prostate Cancer. PLoS ONE 9(3): e91817. doi:10.1371/journal.pone.0091817
Editor:Allen Gao, UC Davis Comprehensive Cancer Center, United States of America ReceivedDecember 31, 2013;AcceptedFebruary 13, 2014;PublishedMarch 14, 2014
Copyright:ß2014 Zhu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding:This study was supported in parts by a grant from the National Natural Science Foundation of China (81041102) and Scientific Research Preferred Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests:The authors have declared that no competing interests exist. * E-mail: zsha@zzu.edu.cn
Introduction
Prostate cancer (PCa) is the most common cancer which represents a great threat to men’s health. Androgen deprivation therapy (ADT) involving surgical or chemical castration is the standard treatment for patients with advanced PCa [1]. However, most patients will become refractory to androgen deprivation and ultimately progress with castration-resistant diseases [2], usually within 12–24 months from initiation of hormonal therapy [3]. The emergence of aggressive castration-resistant clones during ADT is rationale for taxane-based therapy, which is the only chemother-apy class to show a survival benefit in metastatic castration resistant prostate cancer (CRPC) [4,5].
Docetaxel (DTX) is the first-line chemotherapeutic option for symptomatic CRPC patients who are candidates for chemother-apy [6], which enhances the overall response, clinical remission of the prostate cancer patients [7]. DTX treatment increases Bcl-2 phosphorylation, down-regulates Bcl-XL protein levels, induces p53 and thus results in apoptosis [8,9]. In addition, DTX was reported to exert antiangiogenic effects [10]. It reminds us of the early evidence that taxotere could inhibit the proliferation of human umbilical vein endothelial cell proliferation through
inhibition of VEGF secretion [11]. Therefore, we investigated VEGFA secretion before and after treatment with various agents. However, cytotoxicities especially peripheral neurotoxicity and hematopoietic side-effects are significant and inevitable progres-sion occurs after DTX treatment [12,13]. Resistance can develop through a variety of mechanisms include inhibition of apoptosis and activation of the extracellular signal-related pI3 kinase/Akt survival pathways with the development of metastasis [14]. Due to resistance, it often fails to cure patients, therefore, it is important to identify better or alternative therapeutic strategies that reverse chemotherapy resistance and enhance sensitivity to docetaxel-based chemotherapy drugs.
dynamic expression can vary depending on the phenotypic feature of each cell line [17]. The synthetic somatostatin analog octreotide (OCT) has been extensively studied as one of the SST analogs and accumulating evidence supports its antitumor activity in cancer therapy [18,19]. OCT has been approved by FDA to be used as a standard of care for the medical treatment in various types of cancers. It has a highly improved stability compared with natural somatostatin, allowing for long-term treatment [20].
Combing different anticancer agents is a reasonable strategy with which to obtain a potent cytotoxic effect in cancer cells. In our study, we determined the effect of combined treatment of DTX and OCT on the profile of genes expression associated with apoptosis, angiogenesis and drug-tolerance in DU-145 cells. The results presented herein showed that the association of DTX and OCT provided a more than additive apoptotic response. This novel drug combination was more efficient at inducing apoptosis and reducing migration than either drug alone. These results provide an overall understanding of the clinical strategies of prostate cancer and drug resistance reversal.
Materials and Methods
Cells and cell culture
DU145 prostate cancer cells and PC3 cells were provided kindly as a gift by Prof. Peter Nelson (Fred Hutchinson Cancer Research Center). They are purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, USA) that was supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 1% penicillin/streptomycin at 37uC in a humidified atmosphere with 5% CO2. Confluent cells were passaged with trypsin-EDTA (0.05% trypsin and 0.53 mM tetrasodium EDTA) and harvested to prepare mRNA as described below.
Treatment and cell viability assay
DU145 prostate cancer cells were seeded in 96-well plates in quintuplicate with Dulbecco’s basal medium plus 10% fetal bovine serum and maintained in culture for 24 h. The cells were treated with DTX (5, 10, 20, 50 or 100 nM) and OCT (10, 102, 103, 104, 105nM), used alone or in simultaneous combinations for 24 h, 48 h and 72 h, respectively. At the end of incubations, cell medium was removed and 100mL/well of MTT solution (0.5 mg/mL in PBS) were added. Then, plates were incubated for 3 h (at 37uC, protected from light). After incubation at 37uC, 5% CO2 for 4 h, the supernatants were removed carefully, and 150ml of DMSO was added to each well. The cells were then shocked for 10 min in the dark. Absorbance was measured at 450 nm in a Microplate Reader (Bio-Rad 680). Analysis of the obtained results was done using GraphPad Prism 4 computer program to evaluate cell proliferation rate and cytostatic rate. Untreated cells were used as controls.
RNA and DNA extraction
Total RNAs from cultured cells were extracted using Trizol reagent (Takara, Dalian, China) according to the manufacturer’s instructions and their concentrations were determined by a spectrophotometer (NanoDrop, Nyxor Biotech). Genomic DNAs from cultured cells were extracted using DNeasy Blood & Tissue Kits (Qiagen) and their concentrations were measured with the NanoDrop Spectrophotometer. All the processes were carried out according to the manufacturers’ instructions.
RT-PCR and Q-RT-PCR
Quantitative real-time PCR was performed with the QPK-201 SYBR Green master mix (Toyobo, Osaka, Japan) and the ABI 7300 system from Applied Biosystems. The primers used in the study were obtained from Invitrogen (Beijing, China) and presented in Table 1. Thermocycling parameters included a RT step at 50uC for 20 min, followed by a DNA polymerase activation step at 95uC for 2 min and 50 PCR cycles (95uC for 20 s, 60uC for 30 s). All reactions were conducted in triplicate. The fold-change in differential expression for each gene was calculated using the comparative CTmethod (also known as the 22
DDCT
method) as described by Lu et al [21].
Flow cytometric evaluation of annexin V-FITC/PI-stained cells
Cancer cells were grown on 6-well plates for 24 h, treated with DTX (20 nM) and OCT (103nM) alone or in simultaneous combination for additional 48 h. To detect apoptotic events, cells were washed twice with PBS and the pellet was re-suspended in 16Annexin binding buffer at a concentration of 106cells/ml. Then, 100ml of the cell suspension was transferred to 5 ml culture tube. Next, 5ml of the Annexin V-FITC and 5ml of Propidium Iodide were added to each test, and samples were incubated for 15 min at room temperature in darkness. After this time, 400ml of 16Annexin binding buffer was added to each tube and cells were analyzed using a BD FacsCanto flow cytometer (BD Biosciences). Each experiment included the control samples cells stained with Annexin FITC (no PI) and cells stained with PI (no Annexin V-FITC). Viable cells were Annexin V-FITC- and PI-negative, cells in early apoptosis were Annexin V-FITC-positive and PI-negative and cells in late apoptosis or dead were positive both for Annexin-FITC and PI. Results were represented as fold changes in relative mean ratios between cells untreated as compared with the different treatment modalities. Each experiment was performed in tripli-cate.
Wound healing assay
The scratch assay was performed to assess the influence of DTX and OCT on prostate cancer cell motility alone and in combination. DU145 cells were seeded in a high density at 6-well plates and grown for 24 h. The medium was removed and replaced with medium containing DTX and/or OCT for another 24 h. The monolayer cells were physically wounded by scratching the surface with a pipette tip (1000ml) as uniformly and straight as
Table 1.Primers used for RT-qPCR analysis.
Objective genes Primer sequences
CASP3 Forward: 59-CTCATACCTGTGGCTGTGTATC-39
Reverse: 59-GCTCCTTTTGCTGTGATCTTC-39
ACBC1 Forward: 59-GTCGGAATGGATCTTGAAGGG-39
Reverse: 59-ACATCAAACCAGCCTATCTCC-39
CASP9 Forward: 59-AGAGATTCGCAAACCAGAGG-39
Reverse: 59-CAAGATAAGGCAGGGTGAGG-39
VEGFA Forward: 59-CCGAAACCATGAACTTTCTGC-39
Reverse: 59-CCTTTCCCTTTCCTCGAACTG-39
b-actin Forward: 59-AAGGGCCATCCACAGTCTTC
Reverse: 59-AGAAGGCTGGGGCTCATTG
possible. The images of cells invading the scratch were captured at indicated time points (0, 4, 6, 8 and 24 hrs) using phase contrast microscopy (IX 70 Olympus Optical Co., Germany). The pictures were evaluated by measuring the difference in the area of the wounds with a Leica image analysis system (Leica, Mannheim, Germany) and migration rate expressed as percentage of scratch closure was calculated as follows: % of scratch closure = a2b/a, where (a) is a distance between edges of the wound, and (b) is the distance which remained cell-free during cell migration to close the wound. The experiments were repeated in triplicate wells at least two times.
Statistical analysis
The data from the experiments were analyzed using the SPSS 18.0 software and expressed in the form of the mean6SD. The two-sided Student’s t-test was used to analyze the differences in the experiments. The p-value ,0.05 was regarded as significant whereas p-value,0.01 or,0.001 as highly significant.
Results
Apoptotic properties of DU145 cells treated by DTX alone, OCT alone and their combination for 48 h
In the initial study, the growth inhibitory effect of DTX, OCT and the combination of them on prostate cancer cells DU145 was examined. Following these agents treatment, parts of the cells start to shrank, inter-cell spacing became larger and cytoplasmic particle deposition was observed in the DTX and OCT treated cells. This effect was much more powerful in the combination treatment group than in the individual treatment groups. There were more floating and foaming cells displayed morphological features of apoptosis in the combination treatment groups and the increase in the treatment concentration enhanced the apoptotic appearances. Fig. 1 shows the cell morphology of different concentrations of DTX and/or OCT treatment for 48 h. In addition, as the treatment time progressed, cell growth became slow and there were increased numbers of floating cells can be observed in the medium.
Growth inhibition by DTX and OCT alone respectively The growth inhibitory effect of DTX and OCT was examined respectively. It is obvious that each of the two agents displayed
anti-proliferation effect on DU145 cells (Fig. 2A and 2B). Both DTX and OCT decreased cell proliferation in a time and dose dependent manner. As shown in Fig. 2C, compared with untreated controls, there were 14%, 31% and 43% inhibition in the proliferation of DU145 cells exposed to 10 nM of DTX at 24 h, 48 h and 72 h. As for OCT (Fig. 2D), compared with the control, the there was a 10%, 18% and 29% inhibition in the proliferation of DU145 cells exposed to 100 nM of the drug at 24 h, 48 h, and 72 h. Data showed that the highest cytotoxicity was at 72 h and IC50values of DTX (Fig. 2A) and OCT (Fig. 2B) were calculated from cell proliferation plots respectively. DTX exhibited a stronger cytotoxity with the lower IC50with a value of 10.78463.122 nM than the OCT showed a weaker inhibition effect with IC50being 2.34260.262mM.
DTX along with OCT produces synergistic inhibition of growth in human DU145 cells
To assess the possible synergistic effects of DTX-OCT combined on the growth of the cancer cells, DU145 cells were exposed to different concentrations of DXT and OCT combined for 72 h (Fig. 3B). The synergistic effect of DTX and OCT on DU145 cells proliferation was observed. The percentages of cell growth inhibition induced by DTX in combination with OCT were greater than each single agent alone. At the concentration of 103nM, OCT significantly reduced the IC50 value (P,0.05) and promoted apoptosis (P,0.05) in the DU145 cells in response to DTX. As shown in Fig. 3A, 10 nM DXT and 102nM OCT result in a 39% and 14% inhibition in proliferation in DU145 cells, respectively while both combined at the same doses caused a 68% decrease in cell proliferation compared with untreated control, which indicated a strong synergy.
VEGFA, CASP3, CASP9 and ABCB1 mRNA expression in DU145 cells
To explore the potential mechanism by which DTX plus OCT induces apoptotic cell death, the expression of genes involved in apoptotic signal, cell resistance to DTX and angiogenesis were evaluated by RT-PCR and qRT-PCR following 48 h (Fig. S3) and 72 h of treatment. Compared to the control, 10 nM DTX and 100 nM OCT, the two agents combination group markedly decreased VEGFA mRNA expression (Fig. 4A) (P,0.01). As for CASP9 and CASP3, the basal mRNA level of CASP9 and CASP3
Figure 1. Comparison of the morphology of DU145 cells treated with DTX, OCT alone and the two-drug combination for 48 h.A. Untreated cells; B. 1 nM DTX; C. 100 nM OCT; D. 10 nM DTX; E. Two-drug combination of 10 nM DTX and 100 nM OCT; F. 100 nM DTX.
were low but detectable and the mRNA expression was increased significantly as demonstrated in Fig. 4B and Fig. 4C (P,0.01). The protein expression level of the two caspase genes was also examined and consistent with the result of qRT-PCR (Fig. S4). In addition, the expression of ABCB1, which is recognized as a gene associated with drug resistance [22], was not affected in the
treatment groups (Fig. 4D). These results imply that the anti-tumor effect of DTX in combination with OCT might be involved in promoting apoptosis associated with high expression of caspase 9 and caspase 3.
Figure 2. Inhibition of cell growth by different concentrations of DTX (A), OCT (B) after 72 hours incubation and in a time- and dose-dependent manner both of them respectively (C and D) on DU145 proliferation.Cell viability was determined by MTT assay and expressed as a percentage of the control value (mean6SEM of three experiments done in triplicate); error bars = SEM (n = 6).
doi:10.1371/journal.pone.0091817.g002
Figure 3. Effect of DTX alone, synergistic effect of DTX in combination with 100 nM OCT (Fig. 3A) and various concentrations of two agents (Fig. 3B) on DU145 cells proliferation following 72 h of treatment.Results are the percentage of control value obtained with untreated cells (mean6SEM of three experiments done in triplicate). (**) Proliferation was significantly decreased (p,0.001); error bars = SEM.
Effects of DTX, OCT alone and two-drug combination treatment on DU145 cells apoptosis
According to the qRT-PCR results, the experiments were divided into four treatment groups, including the control, DTX (10 nM), OCT (100 nM) and the two-drug combination groups (Fig. 5). Following treatment for 48 h, an apoptosis test was carried
out by flow cytometry according to the instructions provided in the Annexin V-FITC/PI staining kit (Fig. 5A). In contrast to the control group, apoptosis was induced in both the DTX and OCT groups (P,0.05). Consistent with previous observations, the proportion of apoptotic cells was markedly increased by applica-tion of DTX plus OCT as compared with DTX or OCT Figure 4. Effects of DTX, OCT alone and the combination of DTX/OCT on the mRNA expression of VEGFA, caspase 9, caspase 3 and ABCB1 in DU145 cells.M: Marker, The expression levels of objective genes were examined by RTPCR (A, B, C and D) and quantitative realtime -PCR (B, D, F and H) respectively, using cells treated with the test drugs for 72 hours. The expression level of each mRNA was normalized to the level of b-actin mRNA. Values represent the means6SD of triplicate analyses (*p,0.05, **p,0.01).
treatment alone (Fig. 5B). These results indicate that the combination treatment enhanced apoptotic cell death in DU145 cells.
Effect of DTX, OCT alone and two-drug combination on the migration of the DU145 cells
To detect the effect of the two-drug combination on the migration of DU145 cells, a wound healing assay was performed following 24 h treatment. The results of the wound healing assays showed that either of the drug alone have the inhibitory effect on cells migration. But the cells in the combination group migrated more slowly than those in the drug alone and untreated control groups (Fig. 6A). Compared with the control, the relative migrated distance of the cells in the DTX, OCT and DTX/OCT combined groups (Fig. 6B), respectively were 76.5610.21, 53.2612.13, 79.4613.04 (P,0.05). The cells incubated in the control group migrated across an area that was markedly larger than that of the cells incubated in the drug-combined group, indicating that DTX in combination with OCT inhibited the migration of the DU145 cells.
Discussion
Microtubule-targeting based chemotherapy is the most widely used method in the treatment of prostate cancer [23]. However, long-term treatment often results in side effects and chemotherapy resistance, usually contributing to the variable clinical outcomes among patients with seemingly similar tumor types. In an attempt to overcome drug resistance, we decided to investigate the effective
cytotoxic and apoptotic concentrations of the microtubule-targeting drug docetaxel (DTX) and somatostatin analogue octreotide (OCT) and their synergistic effect of the two drugs in castration resistant prostate cancer cell line DU145.
In this study, we evaluated the combined effect of DTX and OCT against prostate cancer DU145 and PC3 cells (Fig. S1). The combination of DTX with OCT induced a greater decrease in cell viability than either agent alone. OCT reduced the IC50value of DTX in DU145 and PC3 cells in a dose-dependent manner, inhibited PCa cells proliferation (Fig. S2), increased DTX sensitivity, cellular cytotoxic and apoptosis. Although we found that PC3 cells showed more sensitive to DTX than DU145 cells which consistent with the study in vivo (Fig. S5). These results suggest that OCT synergistically potentiates the efficacy of microtubule-targeting agents in PCa cells in vitro. These consistent with the recent study by Lattanzio et al. docetaxel combined with octreotide synergistically enhanced the effect of combined treatment in a particularly docetaxel-resistant PC-3 cell line [24]. However, we found either of the two drugs alone stimulates proliferation of DU145 cells in a lower concentration of DTX,1 nM or OCT,10 nM respectively, which was not observed in their combined group.
The anti-proliferative effect of DTX and OCT may be due to either cell necrosis or cell apoptosis. Therefore we investigated the apoptotic effect of DTX, OCT alone and their combination treatment in DU145 cells. Results showed that the combination of DTX with OCT decreased cell viability and increase caspase 9/3 activity to a greater extent than either single agent alone in a time dependent manner (Fig. S4). Compared with the control there is Figure 5. Effect of DTX, OCT alone and two-drug combination treatment on DU145 cells apoptosis.Data are mean6SEM of two independent experiments performed in triplicate. ***p,0.000 vs. control.
obvious increase in caspase 3 expression particularly in the OCT alone and two-drug combined treatment groups. This is consistent with the report showed that OCT increased the caspase 3 mRNA expression and induced cell apoptosis in HepG2 cells [18]. It was indicated that apoptotic programme of cell death was fired by activating the initiator caspase-8, inducing mitochondrial outer membrane permeabilization (MOMP), mitochondrial fragmenta-tion, and ultimately activation of the downstream proteases caspase-9 and -3. MOMP induced the exit of cytochrome c, the formation of apoptosome and ultimately activation of caspase-9 [25]. In the present study, Caspase 9 activity was significantly increased after treatment of DU145 cells after the combination of DTX and OCT treatment, implicating the MOMP and mitochondrial fragmentation induction, resulting in the observed activation of apoptosis in the DU145 cell line.
VEGF signaling promotes angiogenesis by stimulating vascular endothelial cell proliferation, migration and tube formation and markedly increases vascular permeability [26]. The VEGF family includes five members, VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF) and VEGF-A is known for its key roles in tumor-related angiogenesis [27]. Therefore, we investigated the effect of OCT, DTX and the combination of the two drugs on VEGFA expression in DU145 cells. Study showed that the combination of DTX and OCT exert a great
inhibitory effect on VEGFA mRNA expression but had a moderate effect by DTX alone treatment. Report demonstrated that VEGF expression was significantly suppressed in glioma cells by OCT dose-dependently. However, OCT had little effect in the microenvironment of hypoxia-induced VEGF production in vivo [28].
In the present study we demonstrated a significant decrease in cell migration in vitro after exposure to the two-drug combination treatment. Study suggests that DTX in combination with OCT inhibits DU145 proliferation or migration through inhibition of release of the peptide VEGFA and subsequently results in the inhibition of the cell migration. Study reported that the increase of VEGF/VEGFR interaction enhances the migration of colon cancer cells [29]. However, some inconsistent study results were discovered. For example, in Hepatic ischemic-reperfusion injury model, OCT showed the inhibitory effect on hepatocellular apoptosis and attenuated hepatocyte damage caused by ischemic-reperfusion [30]. Short-term application of OCT could induce OCT receptor SSTR2 desensitization and internalization, which inhibited the apoptotic effect of OCT in liver cancer cells [31]. These contrary results may be due to the different cell characteristics, cell microenvironment, expression of the receptors and OCT treatment concentration.
Figure 6. Effects of DTX, OCT alone and two-drug combination on DU145 cells migration.(A) Wound healing assay to determine the effect of indicated drugs on DU145 cells migration. (B) The summarized migration ratio (%) following 72 h treatment measured by wound healing assay. Each column represents the mean6SEM. **p,0.01, vs. control.
It is reported that ABCB1 is expressed in over fifty percent of drug-resistant cancers and recognized as a main mechanism underlying drug resistance [32]. ABCB1 expression was up-regulated after chemotherapy in various tumors as a generalized stress response [33,34]. In our study no significant difference was noted among the various groups treated with combined DTX and OCT, each single agent alone and the untreatment control. High expression of ABCB1 gene before and after these drugs treatment indicates a DTX and/or OCT drug resistant cell line of prostate cancer DU145 cells which has been observed in our previous study by mouse xenograft model in vivo (data not published).
These data suggest that combined treatment of DTX and OCT in DU145 and PC3 cells exerted a greater inhibition of tumor cell proliferation and markedly induced apoptosis, which may be related to the up-regulation of caspase 9/3 activity and decreased expression of VEGFA in vitro. Although our results need to be extended to in vivo models in future studies, they raise the intriguing possibility that targeting microtubules with a taxane combined a somatostain analogue may be a useful approach to improving outcomes in prostate cancer.
Supporting Information
Figure S1 Effect of DTX alone, synergistic effect of DTX in combination with 100 nM OCT on PC3 cells prolifer-ation following 48 h (A) and 72 h (B) of treatment.Results are the percentage of control value obtained with untreated cells (mean 6 SEM of three experiments done in triplicate). (**) Proliferation was significantly decreased (p,0.001); error bars = -SEM.
(TIF)
Figure S2 Synergistic effect of DTX in combination with OCT on DU145 cells proliferation following 48 h of treatment.Results are the percentage of control value obtained
with untreated cells (mean6SEM of three experiments done in triplicate). (**) Proliferation was significantly decreased (p,0.001); error bars = SEM.
(TIF)
Figure S3 Effects of DTX alone, OCT alone and the combination of DTX/OCT on the expression of VEGFA, caspase 9, caspase 3 and ABCB1 in DU145 cells by qRT– PCR respectively, using cells following 48 h of treat-ment.The expression level of each mRNA was normalized to the level of b-actin. Values represent the means6SD of triplicate analyses (*p,0.05, **p,0.01).
(TIF)
Figure S4 Detection of caspase 3, and caspase 9 expression in DU145 cells by western blot (A). Results show that significantly increased caspase 3 and caspase 9 expression levels in DTX alone and in combination with OCT following 48 h treatment. (B) Densitometric analysis was per-formed using Kodak one-dimensional image analysis software. (p,0.01).
(TIF)
Figure S5 Growth curves of prostate cancer xenograft PC3 (A) and DU145 (B) in control mice, castrated mice and mice treated with docetaxel including two groups of 10 mg/kg and 20 mg/kg docetaxel treatment. Tumor measurement starts from the drug treatment (p,0.015). (n = 5).
(TIF)
Author Contributions
Conceived and designed the experiments: SZ YD. Performed the experiments: JAO MHZ YT. Analyzed the data: SQL YT. Contributed reagents/materials/analysis tools: SZ YD SQL. Wrote the paper: SZ YD.
References
1. Stattin P, Kaaks R (2003) Prostate cancer, insulin, and androgen deprivation therapy. Br J Cancer 3;89(9):1814–1815.
2. Shen HC, Balk SP (2009) Development of androgen receptor antagonists with promising activity in castration-resistant prostate cancer. Cancer Cell 2;15(6): 461–463.
3. Sua´rez C, Morales-Barrera R, Ramos V, Nu´n˜ez I, Valverde C, et al. (2013) Role of Immunotherapy in Castration-Resistant Prostate Cancer (CRPC). BJU Int. 12, doi: 10.1111/bju.12110.
4. Nakabayashi M, Oh WK, Jacobus S, Regan MM, Taplin ME, et al. (2010) Activity of ketoconazole after taxane-based chemotherapy in castration-resistant prostate cancer. BJU Int 105(10):1392–1396.
5. Ploussard G, Terry S, Maille´ P, Allory Y, Sirab N, et al. (2010) Class III beta-tubulin expression predicts prostate tumor aggressiveness and patient response to docetaxel-based chemotherapy. Cancer Res 15;70(22):9253–9264.
6. Atmaca A, Al-Batran SE, Werner D, Pauligk C, Gu¨ner T, et al. (2013) A randomised multicentre phase II study with cisplatin/docetaxel vs oxaliplatin/ docetaxel as first-line therapy in patients with advanced or metastatic non-small cell lung cancer. Br J Cancer 5;108(2):265–270.
7. van Soest RJ, de Morre´e ES, Shen L, Tannock IF, Eisenberger MA, et al. (2013) Initial Biopsy Gleason Score as a Predictive Marker for Survival Benefit in Patients with Castration-resistantProstate Cancer Treated with Docetaxel: Data from the TAX327 Study. Eur Urol. Aug 11.
8. Parrondo R, de Las Pozas A, Reiner T, Perez-Stable C (2013) ABT-737, a small molecule Bcl-2/Bcl-xL antagonist, increases antimitotic-mediated apoptosis in human prostate cancer cells. Peer J 12;1:e144. doi: 10.7717/peerj.144. 9. Caraglia M, Giuberti G, Marra M, Di Gennaro E, Facchini G, et al. (2005)
Docetaxel induces p53-dependent apoptosis and synergizes with farnesyl transferase inhibitor r115777 in human epithelial cancer cells. Front Biosci 1;10:2566–75.
10. Wilson C, Scullin P, Worthington J, Seaton A, Maxwell P, et al. (2008) Dexamethasone potentiates the antiangiogenic activity of docetaxel in castration-resistant prostate cancer. Br J Cancer 16;99(12):2054–2064. 11. Avramis IA, Kwock R, Avramis VI (2001) Taxotere and vincristine inhibit the
secretion of the angiogenesis inducing vascular endothelial growth factor (VEGF) by wild-type and drug-resistant human leukemia T-cell lines. Anticancer Res 21(4A):2281–2286.
12. Maggioni D, Nicolini G, Chiorazzi A, Meregalli C, Cavaletti G, et al. (2010) Different effects of erythropoietin in cisplatin- and docetaxel-induced neurotox-icity: an in vitro study. J Neurosci Res 1;88(14):3171–3179.
13. Vainas O, Ariad S, Amir O, Mermershtain W, Vainstein V, et al. (2012) Personalising docetaxel and G-CSF schedules in cancer patients by a clinically validated computational model. Br J Cancer 21;107(5):814–822.
14. Jiang CC, Yang F, Thorne RF, Zhu BK, Hersey P, et al. (2009) Human melanoma cells under endoplasmic reticulum stress acquire resistance to microtubule- targeting drugs through XBP-1-mediated activation of Akt. Neoplasia 11(5):436–447.
15. Bousquet C, Guillermet-Guibert J, Saint-Laurent N, Archer-Lahlou E, Lopez F, et al. (2006) Direct binding of p85 to sst2 somatostatin receptor reveals a novel mechanism for inhibiting PI3K pathway. EMBO J. 6;25(17):3943–3954. 16. Appetecchia M, Baldelli R (2010) Somatostatin analogues in the treatment of
gastroenteropancreatic neuroendocrine tumours, current aspects and new perspectives. J Exp Clin Cancer Res 2;29:19.
17. Ruscica M, Magni P, Steffani L, Gatto F, Albertelli M, et al. (2013) Characterization and sub-cellular localization of SS1R, SS2R, and SS5R in human late-stage prostate cancer cells: Effect of mono- and bi-specific somatostatin analogs on cell growth. Mol Cell Endocrinol 5;382(2):860–870. 18. Tsagarakis NJ, Drygiannakis I, Batistakis AG, Kolios G, Kouroumalis EA (2011)
Octreotide induces caspase activation and apoptosis in human hepatoma HepG2 cells. World J Gastroenterol 21;17(3):313–321.
19. Bousquet C, Lasfargues C, Chalabi M, Billah SM, Susini C, et al. (2012) Clinical review: Current scientific rationale for the use of somatostatin analogs and mTOR inhibitors in neuroendocrine tumor therapy. J Clin Endocrinol Metab 97(3):727–737.
20. Casar-Borota O, Heck A, Schulz S, Nesland JM, Ramm-Pettersen J, et al. (2013) Expression of SSTR2a, but not of SSTRs 1, 3, or 5 in Somatotroph Adenomas Assessed by Monoclonal Antibodies Was Reduced by Octreotide and Correlated With the Acute and Long-Term Effects of Octreotide. J Clin Endocrinol Metab 98(11):E1730–9.
22. Kaliszczak M, Patel H, Kroll SH, Carroll L, Smith G, et al. (2013) Development of a cyclin-dependent kinase inhibitor devoid of ABC transporter-dependent drug resistance. Br J Cancer 29;109(9):2356–2367.
23. Engels FK, Sparreboom A, Mathot RA, Verweij J (2005) Potential for improvement of docetaxel-based chemotherapy: a pharmacological review. Br J Cancer 25;93(2):173–177.
24. Lattanzio L, Tonissi F, Monteverde M, Milano G, Merlano MC, et al. (2013) Differential molecular mechanism of docetaxel-octreotide combined treatment according to the docetaxel-resistance status in PC3 prostate cancer cells. Anticancer Drugs 24(2):120–130.
25. Goldberg AA, Beach A, Davies GF, Harkness TA, Leblanc A, et al. (2011) Lithocholic bile acid selectively kills neuroblastoma cells, while sparing normal neuronal cells. Oncotarget 2(10):761–782.
26. Wang X, Chen X, Fang J, Yang C (2013) Overexpression of both VEGF-A and VEGF-C in gastric cancer correlates with prognosis, and silencing of both is effective to inhibit cancer growth. Int J Clin Exp Pathol 6(4):586–597. 27. Perrot-Applanat M, Di Benedetto M (2012) Autocrine functions of VEGF in
breast tumor cells: adhesion, survival, migration and invasion. Cell Adh Migr 6(6):547–553.
28. Mentlein R, Eichler O, Forstreuter F, Held-Feindt J (2001) Somatostatin inhibits the production of vascular endothelial growth factor in human gliom cells. Int J Cancer 15;92(4):545–550.
29. Huang SM, Chen TS, Chiu CM, Chang LK, Liao KF, et al. (2013) GDNF increases cell motility in human colon cancer through VEGF/VEGFR-1 interaction. Endocr Relat Cancer. Oct 28. [Epub ahead of print].
30. Yang J, Sun H, Takacs P, Zhang Y, Liu J, et al. (2013) The effect of octreotide on hepatic ischemia-reperfusion injury in a rabbit model. Transplant Proc 45(6):2433–2438.
31. Schmid HA, Silva AP (2005) Short- and long-term effects of octreotide and SOM230 on GH, IGF-I, ACTH, corticosterone and ghrelin in rats. Endocrinol Invest 28(11 Suppl International):28–35.
32. Januchowski R, Wojtowicz K, Andrzejewska M, Zabel M (2013) Expression of MDR1 and MDR3 gene products in paclitaxel-, doxorubicin- and vincristine-resistant cell lines. Biomed Pharmacother 8. [Epub ahead of print]. 33. Choi JH, Lim HY, Joo HJ, Kim HS, Yi JW, et al. (2002) Expression of multidrug
resistance-associated protein1,P-glycoprotein, and thymidylate synthase in gastric cancer patients treated with 5-fluorouracil and doxorubicin-based adjuvant chemotherapy after curative resection. Br J Cancer 20;86(10):1578– 1585.
