Cucurbitacin D reprograms glucose metabolic network in prostate cancer
Running Title: Cuc D attenuates prostate cancer
Prostate cancer (PrCa) metastasis is the major cause of mortality and morbidity among men. Metastatic PrCa cells are typically adopted for aberrant glucose metabolism. Thus, chemophores that reprogram altered glucose metabolic machinery in cancer cells can be useful agent for the repression of PrCa metastasis. Herein, we report that Cucurbitacin D (Cuc D) effectively inhibits glucose uptake and lactate production in metastatic PrCa cells via rewiring of glucose metabolic network. This metabolic shift by Cuc D was correlated with decreased expression of GLUT1 by its direct binding as suggested by its proficient molecular docking (binding energy -8.5 kcal mol⁻¹). Cuc D treatment also altered the expression of key oncogenic proteins and miR-132 that are known to be involved in glucose metabolism. Cuc D (0.1 to 1 µM) treatment inhibited tumorigenic and metastatic potential of human PrCa cells via inducing apoptosis and cell cycle arrest in G2/M phase. Cuc D treatment also showed inhibition of tumor growth in PrCa xenograft mouse model with concomitant decrease in the expression of GLUT1, PCNA and restoration of miR-132. These results suggest that Cuc D is a novel modulator of glucose metabolism and could be a promising therapeutic modality for the attenuation of PrCa metastasis.
Key words: Cucurbitacin D, PrCa, miRNAs and Glucose metabolism
Prostate cancer (PrCa) is one of the most commonly diagnosed cancer and second leading cause of cancer related deaths among American men in everyday oncology practice . Emergence of castration resistant PrCa (CRPC) and chemo-resistance are the major hurdles for the management of PrCa [2,3]. Various studies have shown that cancer cells are intricately sensitive to metabolic alterations that modifies the metabolic homeostasis [4,5]. To maintain 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  and this glycolytic metabolism displays divergent profile in androgen-sensitive and insensitive PrCa cells . Furthermore, a higher glucose uptake is required for rapid proliferation of androgen-insensitive PrCa cells . 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 studies suggest that GLUT1 appears to be a very important molecular target in PrCa therapy and may leads to successful therapeutic approach for the management of PrCa. 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 of the natural agents have also been shown for their anti-cancer effects via altering glucose metabolism or inhibiting the expression of glucose transporters . Inhibition of glucose uptake reduces cell growth and induces apoptosis in tumor cells . It has been shown that natural compounds that target different tyrosine kinases or ATP binding sites are able to inhibit glucose transporter activity . 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 .
Cucurbitacins are identified as tetracyclic triterpenoids and belongs to Cucurbitaceae family. They are known to have diverse pharmacological activities including anti-inflammatory, antitumor and antimicrobial activities  . Among several derivative, cucurbitacin B, D, E and I have been studied extensively for their strong anticancer activities . Accumulating studies have shown that they are primarily JAK-STAT inhibitors and have shown potent anti-cancer activities   .
In this study, we have evaluated 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.
2.1 Cuc D inhibits growth and induces apoptosis mediated cell death of PrCa cells
In our study, we used androgen-independent PrCa cell lines, PC3 and DU145. To determine the cytotoxic potential of Cuc D in PrCa cells, MTT assay was employed. As shown in Figure 1A, Cuc D treatment dose-dependently inhibited the cell viability of PrCa cells. We next determined the effect of Cuc D on PrCa cell proliferation by xCELLigence and colony formation assays. Results demonstrated that Cuc D treatment (0.1-1.0 µM) inhibited proliferation of DU145 cells as reflected by decrease in baseline cell index (Fig. 1B). Cuc D treatment significantly reduced the number of colony formation in both PC3 (Fig. 1Ci) and DU145 cells (Fig. 1Cii). The effect of Cuc D on colony formation at 25 nM concentration was more significant (P<0.001) in DU145 cells compared to PC3 cells. Since we observed that Cuc D exert potent cytotoxic and growth inhibitory effects, therefore, we further examined the effect of Cuc D on apoptosis induction. PrCa were treated with Cuc D (0.5 µM) for 24 h. and apoptosis inducing effect of Cuc D was analyzed by Annexin V staining followed by Western blot analysis for cleavage in PARP protein. Our results revealed that Cuc D treatment induced apoptosis in DU145 cells as observed by enhanced Annexin V staining compared to vehicle treated cells (Fig. 1D). Western blot results demonstrated that Cuc D treatment dose-dependently increased the protein levels of cleaved PARP in both PC3 (Fig. 1Ei) and DU145 (Fig. 1Eii) cells. These results suggest that Cuc D exhibited potent growth inhibitory and apoptosis inducing abilities in PrCa cells.
2.2 Cuc D arrests cell cycle of PrCa cells in G2/M phase
Cell cycle arrest is one of the attractive target for the management of various types of cancer . Thus, to investigate the effect on cell cycle distribution, PrCa cells were treated with Cuc D (0.5 and 1 µM) and analyzed by flow cytometry. Result showed a dose-dependent increase of Cuc D treated PC3 (Fig. 2 Ai) and DU145 (Fig. 2Aii) cells in G2/M phase as compared to vehicle group. To further understand the molecular mechanism underlying cell cycle arrest by Cuc D, we examined the effect of Cuc D on cell cycle inhibitory proteins (p21and p27). As shown in results, Cuc D treatment (0.1 and 0.5 µM) dose-dependently increased the protein levels of both p21 and p27 in PC3 (Fig. 2Bi) and DU145 (Fig. 2Bii) cells compared to vehicle treated cells.
2.3 Cuc D inhibits the migratory and invasive potential of PrCa cells
PrCa metastasis is one of the major problems for the treatment of PrCa. Thus, to determine whether Cuc D treatment inhibits the migratory potential of PrCa cells, we performed the scratch wound, agarose beads and Boyden chamber assays. Scratch wound assay showed that Cuc D treatment inhibited migration abilities of DU145 cells (Fig. 2C) in dose dependent manner. At 48 h, we observed wound is comparatively more filled in vehicle group than Cuc D treated group. Consistently, agarose beads assay showed that Cuc D inhibited the motility of PrCa cells by performing (Fig. 2D). Furthermore, Boyden chamber assay performed under chemotactic drive also revealed a decrease in migration of PrCa following the Cuc D treatment (Fig 2Ei-ii). Since Cuc D suppressed the motility of PrCa cells, we were further interested to investigate whether Cuc D treatment modulates invasion abilities of PrCa cells. Interestingly, results illustrated that Cuc D (0.5 µM) effectively inhibited invasion of PrCa cells (Fig 2Fi-ii). Taken together, these data suggest that Cuc D suppresses migration and invasive ability of PrCa cells.
2.4 Cuc D treatment decreases glucose metabolism in PrCa cells
Metastatic prostate cancer cells have shown to have higher glucose metabolism . Since, it was observed that Cuc D inhibits metastatic phenotypes of PrCa cells. Therefore, we were interested to investigate whether Cuc D alters the glucose metabolic shift in PrCa cells. Thus, effect of Cuc D on glucose uptake and lactate secretion was determined by using the commercially available kits. It was observed that following the Cuc D treatment, glucose uptake from media was dose dependently inhibited (Fig 3Ai-ii) while a decrease in lactate secretion (3B i-ii) was observed as compared to vehicle group in both PC3 and DU145 cells. Furthermore, the effect of Cuc D on glucose uptake in DU145 cells was further examined by using 2-NBDG, fluorescent probe. As shown in Figure 3C, the intensity of fluorescent signal was dose-dependently decreased in Cuc D treated group as compared to vehicle treated cells. These findings suggest that Cuc D reprograms the metabolic network in PrCa cells
2.5 Cuc D treatment targets the GLUT1 protein via modulating miR-132 expression
We next determined the effect of Cuc D treatment on protein expression of GLUT1 by Western blot analysis. It was observed that Cuc D treatment suppresses the GLUT1 expression in DU145 (Fig. 4Ai-ii) cells in dose-dependent manner. It is reported that expression of GLUT1 is directly regulated by miR-132 (Fig. 4B) . Therefore, to further understand the underlying mechanisms, we next evaluated the expression of miR-132 following the Cuc D treatment. It was observed that Cuc D treatment showed significant (P<0.05) increase in the expression of miR-132 in PrCa cells (Fig. 4C) as determined by qPCR analysis. Additionally, silencing the miR-132 expression by using inhibitor concomitantly restored the expression of GLUT1 in Cuc D treated DU145 cells (Fig. 4D). These results suggest that Cuc D treatment inhibits the expression of GLUT1 via restoration of miR-132 expression in PrCa cells
2.6 In silico studies displays Cuc D proficiently bind with GLUT1
In order to study the binding pattern of Cuc D with GLUT1,docking analysis were performed. Results showed that ligand Cuc D proficiently binds with GLUT1and binding free energy for this complex was -8.5 kcal mol-1. Figure 4Ei-iishows that Cuc D spatially fit into the binding pocket of GLUT1. Analysis showed that Cuc D interacts with several residues and is involved in hydrogen bonding with THR137, SER80 and ARG153 (Table 4Eii). These results suggest that Cuc D interacts with GLUT1 with both electrostatic (H-bonding) as well as hydrophobic interactions.
2.7 Cuc D inhibits the expression of key signaling components involved in glucose metabolism and cell survival in PrCa
In cancer cells, aberrant activation of PI3K/Akt/mTOR signaling pathway has been associated with enhanced metabolic activities including increased uptake of glucose, amino acid and other nutrients [29-32]. [29-33]AKT is a critical regulator of carbohydrate metabolism and its activation stimulates glycolysis in tumor cells . mTOR is another an important metabolic regulator which is indirectly activated by AKT . Therefore, we investigated whether Cuc D inhibits the expression of key effector molecules of these signaling pathways in PrCa cells. Our results demonstrated that Cuc D treatment inhibits the expression of PI3K, AKT phosphorylation, c-Myc, mTOR phosphorylation and p70S6K in a dose-dependent manner in PC3 (Fig. 5Ai) and DU145 (Fig. 5Aii) cells. Collectively, these results suggest that Cuc D may affect glucose metabolism in PrCa via modulating the aforementioned signaling pathways.
2.8 Cuc D inhibits PrCa cell derived xenograft tumors in athymic nude mice.
To determine whether Cuc D treatment inhibits prostate tumor growth, we developed mouse model using DU145 cells. In this study, a total of 12 athymic nude mice were used to establish PrCa cell-derived xenograft tumors. As the tumor volume measured 100mm3, mice were divided into two groups as described in materials and methods. Results revealed that intra-tumoral injection of Cuc D (1 mg/kg body weight) significantly (p<0.05) inhibited xenograft tumors in athymic nude mice as compared to vehicle treated group (Fig. 6A). Tumors volume were monitored at regular interval of times as shown in figure 6B. Interestingly, Cuc D treated group has demonstrated inhibition of tumor growth over their respective control groups (Fig.6C). Moreover, we determined the effect of Cuc D on the expression of PCNA and GLUT1. Our results showed a marked decrease in the expression of PCNA in Cuc D treated mice xenograft tumors as compared to control tumors (Fig. 6Dii). In addition, there was a decreased in the expression of GLUT1 in Cuc D treated xenograft tumors (Fig. 6Diii). The tumor tissues were further analyzed for the expression of miR-132 by in situ hybridization (ISH). Interestingly, we found increased expression of miR-132 in Cuc D treated excised xenograft tumors when compared with vehicle treatment groups (Fig 6E). These results further confirm anti-tumor efficacy of Cuc D via inhibiting GLUT1 expression and subsequently restoration of miR-132 in in vivo PrCa model.
PrCa is the most commonly diagnosed cancer in men in the United States and its management remains a challenge . Cancer cells are programmed for extensively higher consumption of biofuels to operate their oncogenic machinery. This rewired metabolism is acquired to support their rapid proliferation and metastasis across the body [35,36]. Drug resistance and systemic toxicity are main limiting factors for successful management of PrCa. Therefore, it is imperative to implement 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 GLUT-1 receptor.
Cucurbitacins have been identified potent inhibitor of JAK/STAT pathway . 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 colonogenic 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 is likely to be 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) are critical for the progression of the cell cycle. CDK activity is regulated by CDK inhibitors such as the p21/WAF1 and p27/KIP1 families of proteins. We observed, an upregulation of p21 and p27 proteins in PrCa following Cuc D treatment. These results suggest that Cuc D arrests cell cycle progression in PrCa via modulating cell cycle regulatory proteins.
Furthermore, agents which inhibit migration and invasion of cancer cells could be used for the prevention and treatment of metastatic cancer. Interestingly, we found that non-toxic dose of Cuc D significantly inhibits 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) effectively inhibited invasion of PrCa cells. Our functional experiments show that non-toxic doses of Cuc D significantly inhibit migration and invasiveness of PrCa cells. These findings suggest that Cuc D can also be used to inhibit PrCa cell metastasis.
Accumulative evidence also suggest that glucose deprivation is sufficient to induce growth inhibition and cell death in cancer cells [37-39]. 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 GLUT-1 has been reported in cancer cells but the mechanism of their aberrant expression is not yet clear.  . MiR-132 has been shown to modulate the metabolic flux of PrCa by direct targeting GLUT1 . Strikingly, our findings show that Cuc D treatment restores the expression of tumor suppressor miR-132. Furthermore, we observed using miR-132 inhibitor upregulate GLUT1 expression in Cuc D treated PrCa cells, suggesting Cuc D induces its effect via miR-132 restoration. To gain further insight, we performed molecular docking analysis by Autodock. Our docking results showed that ligand Cuc D proficiently binds with GLUT1and free energy for this complex was -8.5 kcal mol-1. 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 may degrade the GLUT1 protein via binding these residues. However, further studies are warranted to confirm these results.
The PI3K/Akt/mTOR signaling pathways are highly conserve, widely expressed system used by cells to respond to growth factors . Binding of a growth factors (chemokines and cytokines) to its surface receptor activates PI3K, resulting activation of downstream effectors, particularly the serine/threonine kinases Akt and mTOR. Activation of these signaling cascade in cancer cells enhance many of the metabolic activities that support cellular biosynthesis. It permits cells to increase the surface expression of nutrient transported, enabling increased uptake of glucose, amino acid and other nutrients [29-32,42]. Akt increases glycolysis and lactate production and is adequate to induce Warburg effect [33,43,44]. Our results also show the inhibition of PI3K, pAKTSer473, p-mTOR and p70S6 kinase proteins in PrCa cells, which suggests that Cuc D has potential to suppress PI3K/AKT signaling pathways in PrCa cells. These results suggest that Cuc D can reprogram glucose metabolism via targeting these signaling components in PrCa cells.
To further translate our in vitro findings into in vivo, we determined the therapeutic efficacy of Cuc D using an athymic nude mice bearing DU145 cells 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 a dose of 1 mg/kg body weight of Cuc D has potential to inhibit human PrCa cell-derived xenograft tumors without any toxic side effects. Cuc D administration also showed decreased expression of PCNA and GLUT1 proteins in excised xenograft tumor tissues. ISH results further 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.
- Materials and Methods
4.1 Cell culture
The human prostate cancer cells (PC3 and DU145) were kind gift from Dr. Rajesh Singh, Assistant Professor, Morehouse School of Medicine, Atlanta, GA. They procured form 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.) and supplemented with 10% heat-inactivated FBS (Atlanta Biologicals), 1% penicillin, and 1% streptomycin (Gibco BRL). Specific antibodies of Glut-1 (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), p70S6Kinase (cat. no. 9202) were obtained from Cell Signaling Technology Inc. Horseradish peroxidase (HRP)-conjugated anti-mouse (cat. no. 4021) and anti-rabbit (cat. no. 4011) antibodies were acquired from Promega.
Cucurbitacin D was obtained from Dr. Fathi T. Halaweish (SDSU, Brookings, SD). Detailed procedure for synthesis and characterization of Cucurbitacin D was described .
4.2 Cell proliferation assay
The effect of Cuc D (0.1, 0.25, 0.5 and 1µM) on PC3 and DU145 cells proliferation was performed using the MTT assay. After the treatment, 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).
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 .
4.4 Colony formation assay
PrCa (500 cells/per well) were seeded in 6-well plate and Cuc D (25 and 50 nM) treatment was provided for seven days. 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). All of the procedure was followed as described in Vendor’s protocol. 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 . 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 hrs 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), as described earlier . 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 . Data was analyzed by using Modfit software.
4.10 Isolation of RNA and PCR
RNA from prostate cancer cells was isolated using Qiagen kit and quantified using nanodrop instrument 2000 (Thermo Scientific). 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). 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.
4.11 Glucose and lactate assay
Glucose and lactate assays were performed using kits (Cayman Chemicals, #10009582 and #600450) 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.
4.12 Xenograft study
Six-week-old male athymic nude mice were obtained from Jackson Laboratory (Bar Harbor, ME) to generate ectopic xenograft mouse model of PrCa. The 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). A total of 12 mice were used in the study. Briefly, DU145 cells (4 × 106) were dispersed in 100 μL PBS (1X) and 100 μL Matrigel (BD Biosciences) and injected subcutaneously on the dorsal surface of each mouse. The mice were periodically monitored for tumor development and tumor volume was measured from one week after injection using a digital Vernier caliper. When the tumor volume reached ~100 mm3, Cuc D (1 mg/kg) and the respective vehicle control (1X PBS) were administered by intra-tumoral injection three times a week for 5 weeks. The tumor volume was calculated using the ellipsoid volume formula: tumor volume (mm3) = 0.5 × L × W × H, wherein L is length, W is width, and H is height. The tumor was regularly monitored and allowed to grow until the tumor burden of control group mice reached a targeted volume of approximately 1000 mm3. Mice were sacrificed, and their tumors were excised and used for tissue sectioning (5-micron) for histopathology and biochemical analyses.
4.13 Immunohistochemistry (IHC)
IHC analysis for PCNA and GLUT1 proteins were performed on formalin fixed, paraffin embedded xenograft tumors (5-micron sections).
4.14 In situ hybridization for miR-132
The expression of miR-32 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.
4.15 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 . By using standard protocol of AutoDock 4 package, Cuc D was docked into binding site of GLUT1. To deal interactions which exists between GLUT1 and Cuc D, the Lamarckian genetic algorithm (LGA) was applied [23,24]. 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 .
4.16 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. P value < 0.05 was considered as significant.
In summary, this study revealed a novel therapeutic role of Cuc D in rewiring of glucose metabolic network that drive unfavorable tumor microenvironment and inhibits oncogenic signaling pathways in PrCa. Our study offers very promising findings that Cuc D reprograms glucose metabolic network in PrCa and could be a novel chemotherapeutic agent alone or in combination with ongoing chemotherapy regimen for the management of advanced PrCa.
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Legends to the figure:
Figure 1: Effect of Cuc D on cell proliferation, colonogenic potential and apoptosis induction in PrCa cells. (A)Effect of Cuc D on cell viability of PC3 and DU145 cells. Briefly, cells were seeded in 96 well plate and after overnight incubation, treated with indicated concentrations for 48 hrs. Cell viability was assessed by MTT assay. The bar graph represents the percent viable cells compared to vehicle treated cells. Each concentration value is the mean ±SE of triplicate well of each group. (B) Effect of Cuc D on cell proliferation with respect to time was also confirmed by xCELLigence assay. (C) Effect of Cuc D on colony formation of PrCa cells. In brief, 500 cells were seeded in each well of 6 well plates. After 3 days, cells were treated with indicated concertation of Cuc D for 7 days and then media was replaced with complete growth media and colonies were obtained which were further stained with hematoxylin. Photographs were taken by UVP-gel documentation system for PC3 (Ci) and DU145 (Cii). Bar graph represents number of colonies formed in each group of PC3 and DU145 cells. Experiments were repeated in triplicate with similar results. (D) Effect of Cuc D on apoptosis induction of DU145 cells as determined by Annexin V staining. In Brief, 0.5 x 106 cells were seeded in each well of 6 well culture plate. After 24 hrs, cells were treated with indicated concentrations of Cuc D and apoptosis induction was measured by Annexin V staining under fluorescent microscope. Representative images of control and Cuc D treated cells under bright field (BF) (Di) and green fluorescent (GF) (Dii). GF images represent the Annexin V stained cells. (E) Effect of Cuc D on protein levels of early apoptotic biomarker (cleaved PARP) in PC3 (i) and DU145 (ii) cells as determined by Western blot analysis. β-actin was used as internal loading control.
Figure 2: Effect of Cuc D on cell cycle progression, migration and invasive abilities of PrCa cells. (A) Effect of Cuc D on cell cycle distribution in PrCa cells. Cuc D arrests PC3 and DU145 cell cycle in G2/M phase as determined by flow cytometry. (B) Effect of Cuc D on protein levels of cell cycle regulatory proteins (p21 and p27) in PC3 (i) and DU145 (ii) cells as determined by Western blot analysis. (C-E) Effect of Cuc D on migration of PrCa cells as determined by scratch wound, bead assay and Boyden chamber assays. (C) Representative images of migratory DU145 cells in control and treated groups at 0, 48 hrs as determined by scratch wound assay. (D) Representative images of migratory cells (MC) in control and Cuc D treated groups at 0 and 48 hrs as determined by agarose bead assay. (E) Effect of Cuc D on migration of DU145 cells as determined by Boyden chamber assay as described in material and methods. In brief, 18 hrs post-treatment of indicated concentration of Cuc D, migrated cells were fixed, stained and counted. Representative images of migrated control and Cuc D treated DU145 cells (Ei). Bar graph represents the quantification of migrated DU145 cells (Eii). (F) Effect of Cuc D treatment of 18 hrs on invasion of PrCa cells as determined by commercially available kit (BD biosciences) followed by protocol. Representative images of invaded control and Cuc D treated DU145 cells (Fi). Bar graph represents the quantification of DU145 (Fii) cells. Single asterisk (*) denotes the significant value p<0.05.
Figure 3: Effect of Cuc D on glucose uptake and lactate secretion by PrCa cells. (A) Effect of Cuc D on glucose uptake by PrCa cells as determined by glucose colorimetric assay kit (Cayman Chemicals) according to manufacturer’s instruction. Bar graphs represent the unused glucose in PC3 (Ai) and DU145 (Aii) cells 24 hrs post-treatment of indicated concentrations of Cuc D. (B) Effect of Cuc D on lactate secretion by PrCa cells as determined by glycolysis cell-based assay kit (Cayman Chemicals) according to manufacturer’s instruction. Bar graphs represent lactate secretion by PC3 (Bi) and DU145 (Bii) cells 24 hrs post-treatment of Cuc D. Asterisk (*) denotes the significant value p<0.05 compared to vehicle treated cells when applied student’s t-test. (C) Effect of Cuc D on glucose uptake as studied by fluorescence microscopy. Photomicrograph represents the uptake of fluorescent probe, 2-NBDG, by DU145 cells. Briefly, cells were treated with Cuc D at indicated concentrations and uptake of 2-NBDG was analyzed by fluorescence microscopy. Green field images represent the fluorescent probe, 2-NBDG.
Figure 4: Molecular mechanism of Cuc D targeting GLUT1 in PrCa cells. (A) Effect of Cuc D on GLUT1 expression in DU145 cells (Ai) as determined by Western blot analysis. Briefly, cells were treated with indicated concentrations of Cuc D for 24 hrs and total cell lysates were prepared and subjected for Western blot analysis for GLUT1 protein level. β-actin was used as an internal loading control. Values shown above the blot are densitometric analysis of GLUT1 blot normalized with β-actin using GelQuant software. (B) Putative miR-132 binding sites in the SLC2A1 3’UTR region of GLUT1. Seven bases (192 through 198) of the SLC2A1 3’UTR are perfect matches (seed sequences) for miR-132 binding. (C) Effect of Cuc D on the expression of miR-132 in PrCa as determined by qPCR analysis. RNU6B was used as an internal control. Asterisk (*) denotes the significant value p<0.05 when applied student’s t-test. (D) Effect of Cuc D on GLUT 1 expression after transfection of the cells with miR-132 inhibitor as determined by Western blot analysis. α-tubulin was used as an internal loading control. (E) Molecular docking studies of Cuc D with GLUT1 as determined by AutoDock 4 package. Cartoon view of Cuc D docked with GLUT1 protein (Ei). Stereo view of GLUT1 binding with Cuc D, showing hydrogen bond donors and acceptors residues around components (Eii). Table depicting the GLUT1 residues interacting with Cuc D (Eiii).
Figure 5: Effect of Cuc D on key effectors involved in glucose metabolism and cell survival in PrCa cells. (A)Effect of Cuc D on protein levels of PI3K, pAKTSer473, c-Myc, phospho-mTOR and P70S6Kinase in PC3 (i) and DU145 (ii) as determined by Western blot analysis. Briefly, cells were treated with indicated concentrations of Cuc D for 24 hrs, total cell lysates were prepared and subjected for Western blot analysis. α-tubulin was used as an internal loading control. Values shown above the blots are densitometric analysis of blots normalized with α-tubulin as done by GelQuant.
Figure 6: Effect of Cuc D on PrCa cell-derived xenograft tumors in athymic nude mice. (A) Representative photograph of athymic nude mice bearing DU145 cells derived xenograft tumors in control (Ai) and Cuc D (Aii) after 7 weeks. A total of 10 mice were used and divided in two groups: control (n=5) and Cuc D (n=5). DU145 cells (4 x 106) were implanted into right flank of each mouse. Cuc D treatment (1 mg/kg) injected 3 days per week after cell implantation and continued until 7 weeks. Control group mice received 0.2 ml PBS. All mice were sacrificed at 7 weeks when control mice tumor reached at targeted volume of ~1000 mm3. (B) Line graph represents regression of xenograft tumors volume in Cuc D treated mice as compared to control group. (C) Bar graph indicates mean of excised tumors weight of control and Cuc D treated mice. Values in bar graph represent mean ± SE of 5 mice tumors in each group. (D) Representative H&E staining images of control and Cuc D treated excised tumors (Di). Effect of Cuc D on expression of PCNA (Dii) and GLUT1 (Diii) in excised tumor tissues of control and Cuc D treated mice as determined by immunohistochemistry using specific antibodies. (E) Effect of Cuc D on the expression of miR-132 in control and treated mice excised tumors as determined by in situ hybridization.