Prostate cancer (PrCa) is one of the most common malignancies and second leading cause of cancer related deaths among American men [1
]. Emergence of castration resistant PrCa (CRPC) and chemo-resistance are some of the major hurdles in managing PrCa [2
]. Accumulating evidence has shown that cancer cells are intricately sensitive to metabolic alterations that modify the metabolic homeostasis [4
]. To maintain a fast growing need for intermediates, cancer cells reprogram their metabolic pathways. Interestingly, it has been reported that early and advanced stages of PrCa have quite different glucose metabolism [6
], and this glycolytic metabolism displays a divergent profile in androgen-sensitive and insensitive PrCa cells [7
]. Furthermore, a higher glucose uptake is required for rapid proliferation of androgen-insensitive PrCa cells [8
]. Glucose transporters (GLUTs) are responsible for glucose uptake in cells by a mechanism of facilitated diffusion. Recent studies have indicated that reprograming of cancer metabolism is a novel therapeutic strategy for PrCa management. These findings suggest that GLUT1 is a very important molecular target for PrCa therapy. Thus, non-toxic agents/pharmacological inhibitors which have potential to modulate glucose metabolism can be useful for PrCa management.
Natural agents have always been appreciated for the treatment of various disease including cancer because of their low or minimal toxicity. Various natural agents have shown their potential therapeutic and preventive effects in in vitro and pre-clinical mouse model systems. Recently, some natural agents have shown their anti-cancer effects via altering glucose metabolism or inhibiting the expression of glucose transporters [9
]. Inhibition of glucose uptake reduces cell growth and induces apoptosis in tumor cells [10
]. It has been shown that natural compounds that target different tyrosine kinases or ATP binding sites are able to inhibit glucose transporter activity [11
]. It has been reported that natural compounds, including flavonoids, are able to modify glucose uptake by regulating GLUT1 expression and/or altering glucose binding to them [12
Cucurbitacins are identified as tetracyclic triterpenoids and belongs to the Cucurbitaceae family. They are known to have diverse pharmacological activities including anti-inflammatory, antitumor and antimicrobial activities [13
]. Among several derivatives, cucurbitacin B, D, E and I have been widely studied for their strong anticancer activities [14
]. Accumulating studies have shown that they are primarily JAK-STAT inhibitors and have shown potent anti-cancer activities [15
In this study, we have evaluated the therapeutic efficacy and underlying molecular mechanisms of Cuc D against PrCa using in vitro and in vivo model systems. The observed effect of Cuc D might be due its effect on altering glucose metabolism, suppression of PI3K/AKT signaling pathways and restoration of miR-132 expression in PrCa cells.
By their nature, cancer cells undergo a higher consumption of biofuels to operate their oncogenic machinery. This rewired metabolism is acquired to support their rapid proliferation and metastasis across the body [27
]. Drug resistance and systemic toxicity are main limiting factors for successful management of PrCa. Therefore, there is an urgent need for biologically safe and effective natural compounds as anti-cancer drugs. Herein, our study delineates the therapeutic
efficacy of Cuc D against PrCa via in reprogramming metabolic shift and molecular interaction with GLUT1 receptor.
Cucurbitacins have been identified as potent inhibitors of the JAK/STAT pathway [15
]. Our findings suggest Cuc D treatment (0–1 µM) induced a marked anti-proliferative effect in PrCa cells (PC3 and DU145). The inhibitory activity of Cuc D on cancer cell growth was also confirmed with a clonogenic assay, indicating that inhibition is irreversible in nature. It also revealed that Cuc D treatment enhanced Annexin V staining and cleavage of PARP protein in PrCa cells, suggesting that inhibitory activity likely occurs via an apoptosis mediated effect. Furthermore, Cuc D arrests the cell cycle progression in G2/M phase. It has been reported that cyclin-dependent kinases (CDKs) activity is regulated by CDK inhibitors such as p21 and p27 families of proteins. We observed, an upregulation of p21 and p27 proteins in PrCa following Cuc D treatment. These findings suggest that Cuc D arrests cell cycle progression in PrCa via modulating cell cycle regulatory proteins.
Furthermore, agents that inhibit the migration and invasion of cancer cells could be used for the prevention and treatment of metastatic cancer. Interestingly, we found that non-toxic doses of Cuc D significantly inhibit the migration of PrCa cells, which represents that Cuc D could be an effective agent to inhibit PrCa cell metastasis. Moreover, our findings demonstrated that Cuc D (0.5 µM) efficiently reduced the invasion of PrCa cells. Our functional experiments show that non-toxic doses of Cuc D significantly decrease migration and invasiveness of PrCa cells. These findings suggest that Cuc D can also be used to inhibit PrCa cell metastasis.
Accumulating evidence also suggests that glucose scarcity is sufficient to induce growth inhibition and cell death in cancer cells [29
]. A number of natural compounds have shown the ability to modify glucose uptake by regulating GLUT1 expression and/or altering glucose binding to them [12
]. Our results demonstrate inhibition of glucose uptake and lactate production by Cuc D in PrCa which provide us a clue that Cuc D may reprogram glucose metabolism to inhibit the growth of metastatic PrCa cells. Notably, western blot analysis showed that Cuc D decreases the expression of GLUT1. Thus, an increase in glucose uptake has been associated mainly with GLUT1 overexpression. Recently, increased expression of GLUT1 has been reported in cancer cells but the mechanism of their aberrant expression is not yet clear [32
]. MiR-132 has been shown to modulate the metabolic flux of PrCa by direct targeting GLUT1 [20
]. Strikingly, our findings show that Cuc D treatment restores the expression of tumor suppressor miR-132. Furthermore, we observed that miR-132 inhibitor upregulates GLUT1 expression in Cuc D treated PrCa cells, suggesting that Cuc D induces its effect via miR-132 restoration. This is in consistent with other studies where natural compounds have been reported to modulate several epigenetic modification processes known to underlie the molecular mechanism involved in tumorigenesis such as non-coding microRNA expression [33
]. To gain further insight, we performed molecular docking analysis by Autodock. Our docking results showed that ligand Cuc D proficiently binds with GLUT1 and free energy for this complex was −8.5 kcal/mol. It showed hydrogen bonding with THR137, SER80 and ARG153 of GLUT1 whereas ALA392, VAL83, IL404, HIS160, GLY408 and TRP388 residues which are responsible for hydrophobic interactions. It may be possible that Cuc D degrades GLUT1 via binding at these residues. However, further studies are warranted to confirm these results.
The PI3K/AKT/mTOR signaling pathways are highly conserved, which are activated by various growth factors in cells [37
]. Activation of these signaling cascades in cancer cells enhance various metabolic activities that are required for cellular biosynthesis [21
]. AKT enhances glycolysis and lactate production and is adequate to induce the Warburg effect [25
]. Our results also show the inhibition of PI3K, pAKTSer473, and p-mTOR proteins in PrCa cells, which suggests that Cuc D has potential to suppress PI3K/AKT signaling pathways in PrCa cells. In a similar study, He et al. (2017) reported that cucurbitacin E inhibited the mTOR expression in human prostate cancer cells [41
]. These results suggest that Cuc D can reprogram glucose metabolism via targeting these signaling components in PrCa cells.
To translate our in vitro findings into in vivo, we determined the therapeutic efficacy of Cuc D using an athymic nude mice model bearing DU145 cell-derived xenograft tumors. This study showed that intra-tumoral administration of Cuc D (1 mg/kg body weight) inhibited xenograft tumors in athymic nude mice. We did not observe any apparent toxicity in any of the Cuc D administered mouse. These results clearly indicate that Cuc D has potential to inhibit human PrCa cell-derived xenograft tumors. This is in consistent with other cucurbitacin analogue which also exhibited the potent anti-tumor activity in other cancer [42
]. Further, it showed down-regulation of PCNA and GLUT1 proteins in excised xenograft tumor tissues. ISH results demonstrated that Cuc D treatment restores the expression of miR-132 in excised xenograft tumors. These results indicated that Cuc D replenished the tumor suppressor miR-132 in vitro as well as in vivo.
4. Materials and Methods
4.1. Cell Culture
The human prostate cancer cells (PC3 and DU145) were a generous donation from Dr. Rajesh Singh (Morehouse School of Medicine, Atlanta, GA, USA). They were procured from ATCC in January 2016. Once received, cells were expanded and stored in liquid nitrogen (passage < 6). For carrying out experiments, cells were thawed and grown for less than 6 months. These cell lines were cultured in RPMI 1640 (HyClone Laboratories, Inc., Logan, UT, USA) and supplemented with 10% heat-inactivated FBS (Atlanta Biologicals, Atlanta, GA, USA), 1% penicillin, and 1% streptomycin (Gibco BRL, Grand Island, NY, USA).
Cucurbitacin D was obtained from Dr. Fathi T. Halaweish (SDSU, Brookings, SD, USA). Detailed procedure for synthesis and characterization of cucurbitacin D was described previously [44
4.2. Cell Proliferation Assay
The effect of Cuc D on PC3 and DU145 cells proliferation was performed using the MTT assay. Briefly, cells were seeded at a density of 5 × 103 cells per well in 96 well plate and allowed to stand for overnight at 37 °C and 5% CO2 incubator. Next day, cells were treated with different concentration of Cuc D (0.1, 0.5 and 1 µM). DMSO was used as vehicle for the treatment of control cells. Fourty eight hours post-treatment, 20 µL of MTT reagent (5 mg/mL) was added in each well and further incubated the plate for 2 h in CO2 incubator. Absorbance was taken after 2 h at 570 nm (SpectraMax M2 spectrophotometer, Molecular Devices, Sunnyvale, CA, USA). The experiment was performed in triplicates. Results were represented as percent viability with respect to the control group.
4.3. Cell Proliferation by xCELLigence Assay
PrCa cells (10,000 cells per well) were seeded in E-plate following the xCELLigence Real Time Cell Analyzer (RTCA) DP instrument manual as provided by the manufacturer [45
]. Average baseline cell index for Cuc D treated cells compared to control cells was calculated.
4.4. Colony Formation Assay
To determine the effect of Cuc D on clonogenic potential of PC3 and DU145 cells, colony formation assay was performed. In brief, 500 cells were seeded per well in 6- well plate and allowed to stand for next three days. The cells were treated with Cuc D with different concentrations (25 and 50 nM) for seven days. DMSO was used as vehicle for the treatment of control cells. Colonies were fixed in methanol, stained with haematoxylin, and counted using UVP 810 software.
4.5. Apoptosis Analysis
The apoptosis inducing effect of Cuc D on PrCa cells was analyzed by Annexin V-FLUOS staining kit (Roche Diagnostic Corp. Indianapolis, IN, USA). All of the procedure was followed as described in vendor’s protocol. Briefly, 60% confluent PrCa cells (PC3 and DU145) cells were treated with Cuc D (0.5 µM) and kept in CO2 incubator at 37 °C for 24 h. Control group cells were treated with DMSO as vehicle. Cells were washed with PBS (1×) and kept in Annexin-V solution for 20 min. Images were captured in bright and green field by fluorescent microscope.
4.6. Cell Migration Assay
Cells motility was performed in vitro scratch wound assay. Briefly, cells were seeded in a 12-wells plate and after 80–90% confluency a standardized wound was made using a 200 µL micropipette tip. Cells were then treated with Cuc D (0.25 and 0.5 µM) and photographed at 0 and 48 h by phase contrast microscopy.
4.7. Agarose Bead Assay
Cells migration was performed by agarose bead- assay as described earlier [46
]. Briefly, cells were mixed into a low melting point agarose solution and drops of suspension were placed onto plates. Cells were treated with Cuc D at 0 and 48 h and the plates were photographed using a phase-contrast microscope.
4.8. Cell Invasion Assay
Cell invasion assay was performed using BD Biocoat Matrigel Invasion Chambers (BD Biosciences, San Jose, CA, USA), as described earlier [3
]. Cells were treated with Cuc D (0.25 µM) followed by incubation for 18 h. Cells were fixed using methanol and were stained with crystal violet. The images were captured at 18 h.
4.9. Cell Cycle Analysis
The effect of Cuc D on cell cycle analysis was performed by flow cytometry as described earlier [17
]. In brief, approximately 70% confluent PrCa cells were treated with Cuc D (0.5 and 1 µM) for 24 h. The cells were trypsinized and washed twice with ice-cold PBS (1×). The cell pellets were resuspended in 50 µL ice cold PBS (1×) and 450 µL cold methanol. The cells were washed twice with ice cold PBS (1×), suspended 500 µL PBS and incubated with 500 µL RNase (20 µg/mL final concentration) at 37 °C for 1 hr. The cells were chilled over ice for 10 min and stained with propidium iodide (50 µg/mL final concentration) for 1 h and analyzed by flow cytometry (BD Accuri C6; Becton Dickinson, Mountain View, CA, USA). Data was analyzed by using Modfit software.
4.10. Western Blot Analysis
The effect of Cuc D on protein expression in prostate cancer cells were determined by Western blot analysis by using specific antibodies of GLUT1 (cat. no. 12939), c-Myc (cat. no. 9402), pAKTser473 (cat. no. 4060), α-tubulin (cat. no. 2144), p21 (cat. no. 2947), p27 (cat. no. 3686), PI3K110 (cat. no. 4249), PARP (cat. no. 9542), PCNA (cat. no. 2586), Phospho-mTOR (Ser2448) (cat. no. 2971) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA) Horseradish peroxidase (HRP)-conjugated anti-mouse (cat. no. 4021) and anti-rabbit (cat. no. 4011) antibodies were acquired from Promega (Madison, WI, USA).
4.11. Isolation of RNA and PCR
RNA from prostate cancer cells was isolated using Qiagen kit and quantified using NanoDrop instrument 2000 (Thermo Scientific, Waltham, MA, USA). To analyze the expression of miR-132 in control and Cuc D treated cells, 100 ng total RNA was reverse transcribed into cDNA using specific primers designed for miRNA analysis (Applied Biosystems, Foster City, CA, USA). The expression of this miRNAs was determined by qRT-PCR using the Taqman PCR master mixture (no AmpErase UNG) and specific primers designed for detection of mature miRNAs (Applied Biosystems). The expression of miRNA was normalized with the expression of endogenous control, RNU6B.
4.12. Glucose and Lactate Assay
Glucose and lactate assays were performed using kits (#10009582 and #600450, Cayman Chemicals, Ann Arbor, MI, USA. Prostate cancer cells were seeded (104 cells/well in 96-well plate) and media collected to measure the amount of lactate after 24 h, and unused glucose levels after 48 h. The samples were analyzed according to the instructions provided in the kit, the readings were recorded, and calculations were done. The fluorescent D-glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) was used as a fluorescent indicator to evaluate glucose update in PrCa cells. Cells were treated with Cuc D (0.25 and 0.5 µM) followed by 2-NBDG and images were captured by fluorescence microscope.
4.13. Xenograft Study
Next, we assessed the anticancer activity of Cuc D in ectopic xenograft mouse model of PrCa. Twelve 6-week old male athymic nude mice (Jackson laboratory, Bar Harbor, ME, USA) were used. Mice were maintained in a pathogen-free environment and all the procedures were carried out as per the protocol approved by the UTHSC Institutional Animal Care and Use Committee (UTHSC-IACUC; protocol number 17-038). Briefly, a mixture of equal volume of DU145 cells (4 × 106) and 100 µL matrigel (BD Biosciences) was injected subcutaneously on the dorsal surface of each mouse. Once the tumor volume reached ~100 mm3, Cuc D (1 mg/kg) and the respective vehicle control (1× PBS) were administered by intra-tumoral injection three times a week for 5 weeks. The tumor volume was periodically measured using the ellipsoid volume formula: tumor volume (mm3) = 0.5 × L × W × H, wherein L is length, W is width, and H is height. Once the endpoint is reached, mice were sacrificed, and their tumors were excised and used for tissue sectioning (5-micron) for histopathology and biochemical analyses.
4.14. Immunohistochemistry (IHC)
The effect of Cuc D was determined on proliferating cell nuclear antigen (PCNA) and GLUT1 proteins in excised tumors by immnunohistochemistry using Biocare kits (Biocare Medical, Concord, CA, USA). Briefly, the tumor tissues were deparaffinized, rehydrated followed by antigen retrieval using a heat induced technique. Samples were incubated overnight for staining with PCNA and GLUT1 antibodies. The slides were counterstained with hematoxylin, followed by mounting with vecta mount (Vector Laboratories, Burlingame, CA, USA) and visualized.
4.15. In Situ Hybridization for miR-132
The expression of miR-132 in FFPE tissues of control and treated xenograft mice was determined by in situ hybridization analysis using Biochain kit (catalog number K2191050; Biochain IsHyb In Situ hybridization kit) according to the manufacturer’s protocol. Briefly, tissues were deparaffinized and fixed in 4% paraformaldehyde and DEPC-PBS for 20 min. They were subjected to digestion using 2× SSC and 0.1% triton X for next 25 min. The tissues were prehybridized with prehybridized solution provided with the kit for 4 h at 48 °C. This followed the hybridization of the slides with hybridization buffer and digoxigenin labelled probe (Exicon, Woburn, MA, USA) at 45 °C overnight. After stringent washing of tissue slide with various grades of SSC, the slides were blocked using 1× blocking solution provided with the kit. This followed the subsequent incubation of tissues overnight with AP-conjugated anti-digoxigenin antibody. Further, the slides were washed for 5 min with 1× alkaline phosphatase buffer twice. The final visualization was carried out with NBT/BCIP (Pierce, Rodkford, IL, USA) followed by nuclear fast red counterstaining. The slides were mounted and analyzed under microscope.
4.16. Molecular Docking
The 2D and 3D structures of Cuc D were retrieved from PubChem. Atomic coordinates for the crystal structure of GLUT1 (PDB ID: 4PYP) were taken from Protein Data Bank (www.rcsb.org
). Other calculations and file preparations were done according to our published protocol [47
]. By using standard protocol of AutoDock 4 package, Cuc D was docked into binding site of GLUT1 [48
]. To deal interactions which exists between GLUT1 and Cuc D, the Lamarckian genetic algorithm (LGA) was applied [49
]. Polar hydrogen atoms were added geometrically. Final docked complexes were optimized, validated and analyzed using “Receptor–Ligand Interactions” modules present in the script section of Discover Studio 4.0. Further to visualize molecular interactions, resultant dock structure files were analyzed by PyMOL [51
4.17. Statistical Analysis
Statistical analysis was performed using an unpaired two-tailed Student t-test and employed to assess the statistical significance between the control and Cuc D treated groups.