Prostate cancer (PC) is the second leading cause of cancer-related deaths among men in Western countries [1
]. A mainstay of treating patients with advanced PC is androgen deprivation therapy (ADT), which involves removal of gonadal sources of testosterone via surgical (bilateral orchiectomy) or medical castration (LHRH agonists or antagonists). ADT induces initial responses in the majority of patients by disrupting androgen receptor (AR)-axis signaling. However, the disease eventually progresses within two years of ADT in most patients despite castrate levels of serum testosterone, resulting in castration resistant prostate cancer (CRPC) [2
]. Such progression is often due to the restoration of androgen-AR signaling under androgen deprived conditions. Therefore, agents targeting either androgen biosynthesis (e.g., abiraterone acetate (Abi)) or AR signaling (e.g., enzalutamide (Enz)), i.e., the so-called androgen receptor-axis-targeted (ARAT) agents, were introduced as a second line therapy in patients with CRPC [3
]. While both Abi and Enz can improve overall survival among responding men, these treatments also eventually fail, resulting in disease progression [4
]. Further, other men with CRPC may be resistant to Abi or Enz de novo [8
]. Studies suggest that resistance to Enz and Abi may in part be due to altered expression of AR and/or AR splice variants in PC cells [10
The finding that patients with diabetes taking metformin, but not other anti-diabetic drugs, have a decreased risk of dying from PC [11
] led to extensive studies on the anti-tumor effects of metformin in PC. Pre-clinical studies demonstrate that metformin can down-regulate AR by disrupting the protein midline-1 (MID1) complex, which otherwise increases AR via enhanced translation [13
]. Other studies demonstrate that metformin can induce apoptotic cell death [14
]. However, as the pharmacologic concentrations used in most of these studies are not readily achievable clinically, the beneficial effects of metformin have been difficult to ascertain in patients with PC.
In addition to caspase-dependent apoptosis, alternative models of programmed cell death (PCD) have been proposed [18
]. Apoptotic cell death can proceed through the activation of both caspase-dependent and -independent pathways. Caspase-independent PCD pathways are important when caspase-mediated routes fail. For caspase-independent PCD, poly (ADP-ribose) polymerase-1 (PARP-1) plays a central role. Normally PARP-1 is involved in the repair of DNA damage induced by a variety of cellular stresses. However, additional functions of PARP-1 have also been revealed (for review, see [21
]). PARP-1 can be cleaved by several ‘suicidal’ proteases. The cleaved PARP-1 fragment containing a DNA binding domain can still bind to DNA but cannot catalyze DNA repair as it lacks the catalytic domain. Thus, the cleaved PARP-1 fragment that binds DNA can act as a dominant-negative inhibitor of PARP-1, inhibiting DNA repair and leading to cell death.
Excessive activation of PARP-1 can also lead to a 10–500-fold increase in poly ADP ribose (PAR) polymer accumulation within the nucleus [24
], which then translocates to the mitochondria to cause a release of mitochondrial apoptosis inducing factor (AIF). AIF release and its subsequent translocation to the nucleus can commit cells to undergo parthanatos [25
]. This phenomenon was first described in neuronal cells undergoing neural degradation and has also been linked to other syndromes connected with specific tissue damage [26
In this paper, we used two human androgen-sensitive PC cell lines, LNCaP and VCaP, to study the role of metformin and ARATs in prostate cancer. We report that in PC cells, metformin, in combination with an inhibitor of androgen biosynthesis (Abi) or an AR targeting agent (Enz), can mediate PARP-1-dependent PCD: a) via enhanced PARP-1 cleavage that is essentially independent of caspase 3 activation, b) via enhanced PARP-1 activation, and c) at lower concentrations than have been observed with metformin in prior studies. This report expands the possible pathways by which metformin-based and ARAT-based targeting strategies could potentially be further developed and enhanced to treat PC.
Although hormonally directed therapies can result in clinically meaningful responses in patients with PC, such therapies are primarily palliative and work for only a limited period in most patients. A major challenge, especially with agents such as abiraterone and enzalutamide, is how to improve their anti-tumor activity while also maintaining treatment safety. Multi-faceted approaches that target not only the hormonal axis but other pathways are being actively evaluated to improve the anti-prostate cancer activity of such approaches.
In addition to its glucose lowering properties, metformin has effects on mitochondrial function and cellular metabolism, including mitigation of hyperinsulinemia and activation of the AMP-kinase pathway, which may contribute to some of its purported anti-proliferative and anti-tumor properties [40
]. Insulin is a growth factor, and hyperinsulinemia present in insulin resistance is associated with lower levels of sex hormone binding globulin, thereby increasing the availability of unbound free androgens. Given that there is wide clinical experience and an established safety profile with metformin, it is a particularly attractive agent to repurpose into combination regimens for anti-cancer therapy, as is being done with some other previously approved drugs [42
] In the present study, we employed two independent well-established cell culture models of human PC, i.e., LNCaP and VCaP cells, to further study and clarify the potential role of metformin in the context of targeting the androgen/AR axis under the backdrop of different molecular characteristics that define these two cell lines. The two cell lines retain relative sensitivity to androgens, and share some but not other underlying biological properties, thus representing some of the heterogeneity seen within the clinical PC disease spectrum. For instance, LNCaP cells express wt p53, mutant (but functional) AR, and mutant PTEN, whereas VCaP cells have abrogated p53 function (due to p53 allelic deletion and missense mutation), express both AR and ARv7, and have wt PTEN
By using several complementary assays, we demonstrate that ARATs, metformin, and ARATs + metformin lead to growth inhibition in both LNCaP and VCaP cells, with greater and statistically significant inhibitory effects noted with the combination treatments. The combination of metformin + ARATs consistently increased the percentage of TB+ (dead) cells compared to untreated- or single agent-treated cells in both cell types. In addition, the proportion of attached mostly TB− (alive) cells was decreased further when metformin was added to ARATs compared to ARAT treatments alone.
Fluorescence-activated cell sorting (FACS) analysis (Table 1
) demonstrated that metformin or metformin + ARAT treatment was associated with increased annexin V staining in LNCaP, but not VCaP cells. Annexin V binds to externalized phosphatidylserine on cell plasma membranes, which is one of the earliest events in several but not all forms of PCD. Interestingly, we also found differential annexin V staining between LNCaP and VCaP cells in response to the potent inducer of PCD, staurosporine; enhanced caspase 3 cleavage occurred in both cell types, but an increase in annexin V staining was observed only in LNCaP cells (Table 1
). Taken together, these data suggest that VCaP cells may recruit a cell death program that shares many but not all features of a canonical PCD pathway typically associated with annexin V staining.
We found ARATs enhanced cleavage of PARP-1 in VCaP but not LNCaP cells (Figure 1
G). However, metformin (1 µM) as a single agent induced PARP-1 cleavage in both cell lines within 4–5 days of treatment (Figure 2
H). Metformin in combination with ARATs is particularly effective in inducing cleavage of PARP-1 (Figure 3
G), which is further underscored by the demonstration of PARP-1 cleavage with the combination even after PARP-1 knockdown with siRNA (Figure 3
H). The combination treatments also enhanced PARP-1 activity, as evidenced by a dramatic increase in PAR levels in the ARAT + metformin treated cells (Figure 3
G). Increased PAR production results in its translocation from the nucleus to the mitochondria causing a release of AIF (cleaved form) from the latter. The cleaved AIF, in turn, recruits DNA endonuclease to the nucleus, which leads to DNA cleavage and a form of PCD termed parthanatos [26
]. Consistent with this paradigm, ARATs + metformin led to increased cAIF in the nuclear fractions of both LNCaP and VCaP cells. A putative mechanism of cell death by cleaved PARP-1 is that it binds to DNA, preventing non-cleaved PARP-1 from accessing the damaged sites and initiating repairs [44
]. Therefore, a relatively small amount of cleaved PARP-1, as observed in our studies, may be enough to block further DNA repair, while the remaining non-cleaved PARP-1 can produce more PAR and thus also contribute to PCD (Figure 3
G). In other studies, we evaluated the potential role of lysosomal proteases in mediating some of the effects of metformin and ARATs in the PC cells since they can amplify the cell death program. Indeed, metformin or metformin, in combination with ARATs, but not ARATs alone, increased lysosomal membrane permeability (LMP) and PARP cleavage in the PC cells, with a partial abrogation of cell death occurring when LMP was inhibited.
ADT and ARATs have proven to be among the most effective anti-PC agents to date clinically, particularly in the castration-sensitive setting. Although this degree of clinical efficacy of ADT/ARATs is perhaps not as adequately reflected in in vitro PC models in that ADT and ARATs primarily induce an anti-proliferative response in cell culture models of androgen responsive PC cells, nevertheless such models provide useful pre-clinical signals that can inform anti-cancer agent activity in the clinical setting. In this regard, our data show that metformin, when added to ARATs, can enhance the anti-proliferative activity of ARATs and increase PCD, and thus are of potential clinical relevance. However, some of the limitations of our study are that it was limited to two androgen-responsive PC cell lines and did not evaluate the combinations in either androgen-insensitive or AR negative PC cells, and was also restricted to in vitro models. Further, the study did not evaluate some of the other effects the combination treatments could have had on the metabolome. The question also remains as to whether adequate levels of metformin to effect anti-tumor responses can be achieved in patients. Some of the failed clinical trials with metformin in non-diabetic cancer patients to date may be due to the lower doses of metformin used in the clinic as compared to pre-clinical studies. During controlled clinical trials, the maximum dose of metformin hydrochloride tablets did not exceed 2550 mg daily, resulting in maximum metformin plasma levels of less than 5 µg/mL (about 30 µM). We tested the effects of metformin at significantly lower doses (1 mM or less) than have been reported by many other investigators in pre-clinical studies (generally 5–30 mM). Although achieving such levels of metformin in plasma remains a challenge, it is transported into cells by the organic cation transporter 1-3 (OCT1-3), which can be highly expressed in prostate tissue and may allow for enhanced intracellular drug accumulation [45
In conclusion, we demonstrate that metformin in combination with ARATs causes enhanced anti-proliferative effects and also induces cell death via pathways other than the canonical apoptotic machinery. The combination results in the recruitment of two PARP-1-dependent cell death pathways, including via enhanced cleavage of PARP-1 and enhanced production of PAR with an associated increase in nuclear cAIF accumulation (Figure 5
). Metformin/ARAT-mediated parthanatos, to our knowledge, has not been described previously in PC. Our study adds to the growing body of evidence regarding the potential range of mechanisms that can mediate anti-tumor effects of metformin in concert with ARATs, including, for instance, the recent demonstration of metformin sensitizing PC cells to enzalutamide via recruitment of STAT3/TGFb signaling [47
]. Many of the initial trials with metformin in PC have evaluated it in the advanced castration resistant setting, a disease state that is generally more refractory to additional therapies compared to castration sensitive disease. Given that metformin enhances the anti-cellular effects of ARATs in androgen-responsive cells, it will be of interest to evaluate prospectively metformin/ARAT-based combinations, particularly in treatment-naïve, castration-sensitive settings of PC. Indeed, large retrospective analysis has demonstrated that diabetic PC patients initiated on ADT who are on metformin have statistically significant better overall and cancer specific survival compared to diabetic PC patients on ADT but not metformin [48
]. Finally, the present study also provides a framework to test whether anti-tumor efficacy can be further improved in PC by incorporating other rationally selected targets into a metformin/ARAT backbone.
4. Materials and Methods
4.1. Materials and Drugs
Acridine orange, metformin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), chloroquine, and E-64d were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Anti-AIF, anti-PAR, and anti-cathepsin G antibodies were purchased from Santa Cruz BioTechnology (Santa Cruz, CA, USA). Antibodies to PARP-1, cleaved PARP-1, Bcl2, Bax, Mcl1, caspase 3, cleaved caspase 3, AR, ARv7, actin and GAPDH, and the horseradish peroxidase labeled secondary antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). All antibodies were used at 1 to 1000 dilution. Enzalutamide (Enz) was purchased from Selleck Chemicals (Houston, TX, USA). Abiraterone acetate (Abi) was a gift from Johnson and Johnson Health Care Systems Inc (New Brunswick, NJ, USA).
4.2. Cell Lines and Culture Conditions
VCaP cells (obtained from ATCC, used at passage numbers less than 40) were grown in DMEM/F12 (1:1) media (BioSource, Grand Island, NY, USA) supplemented with 5% FBS (Biosource), 50 units/mL penicillin, and 50 µg/mL streptomycin. LNCaP cells (obtained from ATCC, used at passage numbers less than 30) were cultured in RPMI1640 medium (BioSource, Grand Island, NY, USA) supplemented with 10% FBS (Biosource), 50 units/mL penicillin, and 50 µg/mL streptomycin. The culture medium was changed every other day. For all cell culture experiments, the cell passage number of wild-type LNCaP cells was 34 or less while that for VCaP cells was 50 or less.
4.3. RNA Preparation and Quantitative RT-PCR (qPCR)
Wild-type cells were plated at a density of 3 × 105
cells/per well in 35 mm petri dishes. The cells were treated singly with metformin (1 mM), Enz (10 µM), or Abi (5 µM), or in specific combinations for 5 days. Total mRNA was extracted using Trizol (Life Technology, Carlsbad, CA, USA) and isolated with RNA mini-prep columns (Qiagen, Valencia, CA). The mRNA was reverse transcribed to cDNA with M-MLV reverse transcriptase (Roche Diagnostics, Basel, Switzerland). Reverse-transcribed cDNA (10–100 ng) was amplified by real-time PCR using IQ SYBR green mix (Bio-Rad, Hercules, CA, USA) and detected via the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad). Each reaction was performed in duplicate. The sequences of AR, Arv7, MID1, and TBP primers are provided in Table 2
. The sequences of Arv7 primers used are in reference [49
4.4. Preparation of Protein Extracts and Western Blot Analysis
We cultured 3 × 105 cells/mL in 35 mm plates. Following the indicated treatments, cells were washed in 1 × PBS; the attached cells were lysed in radioimmune precipitation buffer for 30 min on ice with occasional vortexing. The clarified lysates were separated by 4–12% SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting using the relevant primary antibodies as indicated. The bands were visualized by enhanced chemiluminescence (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA) and quantified by densitometric analysis (Visionworks LS image acquisition and analysis software, UVP, Upland, CA, USA).
4.5. Isolation of Nuclear Fractions
PC cells were seeded in 60 mm dishes and treated with various drugs for 5 days. Nuclear extracts from the cells were prepared using the NE-PER extraction kit (Pierce Thermo Scientific Inc., Rockford, IL, USA) and protein quantified using the BCA assay kit (Pierce Thermo Scientific Inc., Grand Island, NY, USA).
4.6. MTT Assay
MTT assay was performed essentially as described by Mossman [50
]. LNCaP cells were plated in 96-well plates at a density of 3 × 103
cells/well in 100 µL culture medium supplemented with 10% FBS. After two days, cells were treated with DMSO or the agents under study. After the treatment period, 25 µL/well of 5 mg/mL MTT stock solution was added for 3 h, the media subsequently removed, and the resulting formazan crystals dissolved in isopropanol (200 µL/mL) and optical density (OD) determined at 570 nm.
4.7. Trypan Blue Staining Assay
Cell viability was assessed via trypan blue staining. PC cells were plated in 6-well plates at a density of 1 × 106 cells/well. Drugs were added to the plates (in duplicates), and after 5 days of incubation, both floating and attached cells were collected separately, stained with trypan blue, and counted using a hemocytometer.
4.8. Annexin V Detection
Annexin V was detected using the FITC Annexin V Apoptosis Detection kit from BD Biosciences. PC cells were treated with various drugs, washed in 1 × PBS, and the attached cells harvested and incubated with Annexin V/PI according to the manufacturer’s instructions, with cell staining subsequently assessed by flow cytometry.
4.9. Lysosome Staining by Acridine Orange
Cells grown on coverslips and treated with study drugs singly or in various combinations for 5 days were washed with 1 × PBS and then stained with 1 mM acridine orange (Sigma A6014) at 37 °C for 15 min to label acidic lysosomes. Excess acridine orange was washed with 1 × PBS and the cells were examined via a Nikon Eclipse 80i (Nikon Instruments Inc., Melville, NY, USA) wide field fluorescent microscope.
4.10. siRNA Interference
Knock down of cathepsin D, cathepsin G, and PARP-1 mRNA was performed using pre-designed cathepsin D or G siRNA (final concentration 10 nM, Santa Cruz, Dallas, TX, USA), or PARP-1 siRNA (final concentration 10 nM, Cell Signaling Technology). Non-specific siRNA (10 nM, Santa Cruz, Santa Cruz, Dallas, TX, USA) was used as a negative control. Cells were seeded in 35 mm Petri dishes and transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol, then treated with the relevant drugs as indicated 24 h post transfection and incubated for 3 days before cell harvest.
4.11. Drug Combination Index
Drug combination studies were performed according to the methods described by Chou and Talalay [35
]. Three thousand cells/well were seeded in 96-well plates and allowed to attach over 48 h. Drugs were then added at their fixed IC50 ratios at various concentrations as a single agent or in combination, and cells were incubated for 3 days. MTT assays were carried out as described above. The combination index (CI) values at 50%, 75%, and 90% of effective doses and dose reduction index (DRI) values for each drug in the combination were determined using the CalcuSyn 2.1 program [51
4.12. 2-D Clonogenic Assays
We plated 1 × 104
cells per well in 12-well plates. After 48 h, cells were treated with DMSO (control), Abi, Enz, metformin, or combinations and incubated for 10 days. After incubation, media was aspirated, colonies washed with PBS, and fixed with 200-proof ethanol for 30 min. Colonies were then stained with 0.5% crystal violet for 30 min at room temperature, and extra stain washed. The surface area of the wells covered by the colonies was assessed using ImageJ 1.48v software (NIH, Bethesda, MD, USA) (LNCaP cells). CellCounter (https://nghiaho.com/
(accessed on 28 February 2020)) software by Nghia Ho [52
] was used to determine the number of colonies and graphed with GraphPad Prism (v 8.3.1) (VCaP cells). The standard error of mean (SEM) was generated for each treatment from three different wells. A Student’s t
-test was performed to determine p
-values. Three independent experiments were conducted for biological validation of the data.
4.13. Statistical Methods
Student’s t-tests were used for statistical comparisons.