Carbidopa is used with l-DOPA (l-3,4-dihydroxyphenylalanine) to treat Parkinson's disease (PD). PD patients exhibit lower incidence of most cancers including pancreatic cancer, but with the notable exception of melanoma. The decreased cancer incidence is not due to l-DOPA; however, the relevance of Carbidopa to this phenomenon has not been investigated. Here, we tested the hypothesis that Carbidopa, independent of l-DOPA, might elicit an anticancer effect. Carbidopa inhibited pancreatic cancer cell proliferation both in vitro and in vivo. Based on structural similarity with phenylhydrazine, an inhibitor of indoleamine-2,3-dioxygenase-1 (IDO1), we predicted that Carbidopa might also inhibit IDO1, thus providing a molecular basis for its anticancer effect. The inhibitory effect was confirmed using human recombinant IDO1. To demonstrate the inhibition in intact cells, AhR (aryl hydrocarbon receptor) activity was monitored as readout for IDO1-mediated generation of the endogenous AhR agonist kynurenine in pancreatic and liver cancer cells. Surprisingly, Carbidopa did not inhibit but instead potentiated AhR signaling, evident from increased CYP1A1 (cytochrome P450 family 1 subfamily A member 1), CYP1A2, and CYP1B1 expression. In pancreatic and liver cancer cells, Carbidopa promoted AhR nuclear localization. AhR antagonists blocked Carbidopa-dependent activation of AhR signaling. The inhibitory effect on pancreatic cancer cells in vitro and in vivo and the activation of AhR occurred at therapeutic concentrations of Carbidopa. Chromatin immunoprecipitation assay further confirmed that Carbidopa promoted AhR binding to its target gene CYP1A1 leading to its induction. We conclude that Carbidopa is an AhR agonist and suppresses pancreatic cancer. Hence, Carbidopa could potentially be re-purposed to treat pancreatic cancer and possibly other cancers as well.

Introduction

Most epidemiological studies have shown that Parkinson's disease (PD) patients have decreased incidence of most cancers, including pancreatic cancer, with the notable exceptions of melanoma and possibly breast cancer [16]. Most of these patients are treated with a combination-drug regimen consisting of Carbidopa and l-DOPA (l-3,4-dihydroxyphenylalanine). Several studies have investigated the potential effect of l-DOPA on cancer, with an idea that the use of l-DOPA might be responsible for the decreased incidence of most cancers or for the increased incidence of melanoma in patients with PD [710]. But none of these studies showed evidence of involvement of l-DOPA in the decreased incidence of most cancers in association with PD, leaving the puzzling phenomenon unexplained. Interestingly, though Carbidopa is always used in combination with l-DOPA to treat PD, the potential relevance of Carbidopa to decreased cancer risk has never been investigated.

As dopaminergic neurons are selectively decreased in PD, l-DOPA is used to increase dopamine levels in the brain as the treatment strategy. Carbidopa on its own has no therapeutic effect in this disease, but is used in combination with l-DOPA to enhance the potency of the latter. Carbidopa is an analog of l-DOPA, having a methyl group and a hydrazine moiety attached to the α-carbon (Supplementary Figure S1). It is an inhibitor of aromatic amino acid decarboxylase, thus preventing the conversion of l-DOPA into dopamine (Supplementary Figure S1). However, the effect of Carbidopa is restricted to the peripheral tissues and the conversion of l-DOPA into dopamine in the brain is not affected by Carbidopa because of the inability of the latter to cross the blood–brain barrier [11,12]. Dopamine also does not penetrate the blood–brain barrier; therefore, the conversion of l-DOPA into dopamine outside the brain decreases the availability of the drug to the brain. As such, when used in combination with Carbidopa, more l-DOPA enters the brain for subsequent conversion into dopamine; thus, Carbidopa potentiates the therapeutic effect of l-DOPA in PD. At present, Carbidopa by itself is not indicated as a drug for any disease.

The present study was undertaken to test the hypothesis that Carbidopa, always used in combination with l-DOPA, is responsible for the decreased incidence of most cancers in patients with PD. For this hypothesis to be true, Carbidopa on its own must have anticancer effect. Our studies presented here show that this indeed is the case; Carbidopa inhibits the growth and proliferation of pancreatic cancer cell lines in vitro and in vivo. Subsequent studies into the molecular mechanisms reveal that Carbidopa is an agonist for the nuclear receptor AhR (aryl hydrocarbon receptor), and that this function is observed at drug concentrations that are therapeutically relevant to patients with PD. Our studies provide a plausible explanation as to why the cancer incidence is generally decreased in patients with PD. More importantly, our studies demonstrate that Carbidopa is an agonist for AhR and suppresses pancreatic cancer, suggesting that the drug could potentially be re-purposed to treat pancreatic cancer and possibly other cancers. That said, the role of AhR signaling is not consistent in all tumors. It exhibits a pro-oncogenic activity enhancing growth, survival, and migration/invasion (colon cancer, gastric cancer, androgen-independent prostate cancer, urothelial, lung, head and neck cancers, glioma, melanoma, and lymphoma) [13], whereas in others it induces tumor suppression characterized by growth inhibition, apoptosis, and decreased migration/invasion (breast, liver, pancreatic, androgen-dependent prostate, and esophageal cancers) [13]. Nonetheless, AhR is overexpressed in multiple tumors including pancreatic cancer [14], and hence, it is possible that AhR could prove to be an important drug target in some of these tumor types. As such, the idea of re-purposing Carbidopa to treat pancreatic cancer and possibly other cancer is a very attractive strategy. Multiple evidences support the potential of re-purposing an FDA-approved drug to treat cancer. Examples include omeprazole, an FDA-approved proton pump inhibitor, as an anticancer agent in pancreatic cancer [15] and breast cancer [16], leflunomide, an FDA-approved drug for severe rheumatoid arthritis, to inhibit growth in melanoma cells [17], and flutamide, an anti-androgen drug, to suppress hepatocellular carcinoma [18]. Interestingly, all these drugs are also known to be AhR agonists [19]. So, it seems reasonable to state that maybe Carbidopa as an AhR agonist might also prove to be an anticancer agent not just in pancreatic cancer but also in several other cancers.

Materials and methods

Cell culture

Human cell lines, hTERT-HPNE (normal pancreatic epithelial), BxPC-3, Capan-1, and Capan-2 (pancreatic cancer), and HepG2 (liver cancer) cells were all procured from ATCC. These cell lines were used within 10–20 passages. The ATCC has done morphological, cytogenetic, and DNA profile analyses for characterization of these cell lines. AsPC-1, MIA PaCa- 2, and Panc-1 human pancreatic cancer cell lines were obtained from Dr Raj Govindarajan (Ohio State University, Columbus, OH, U.S.A.). HPDE, a human pancreatic ductal epithelial cell line, was kindly provided by Dr Ming Tsao, Ontario Cancer Institute (Toronto, Canada). AsPC-1 and BxPC-3 cells were grown in RPMI-1640 medium, supplemented with 10% FBS (fetal bovine serum), and subcultured at a 1 : 5 ratio. hTERT-HPNE cells were maintained in 75% Dulbecco's Modified Eagle's Medium (DMEM) without glucose plus 25% Medium M3 Base with the following additives: 5% FBS, 5.5 mM d-glucose, 10 ng/ml human recombinant epidermal growth factor, and 750 ng/ml puromycin, and subcultured at a 1 : 4 ratio. MIA PaCa-2 cells were cultured in DMEM, supplemented with 10% FBS and 2.5% horse serum, and subcultured at a 1 : 8 ratio. Capan-2 cells were cultured in McCoy's 5A Modified Medium supplemented with 10% FBS and subcultured at a 1 : 4 ratio. HepG2 cells were cultured in Eagle's Minimum Essential Media supplemented with 10% FBS and subcultured at a 1 : 4 ratio. All media for the above cell lines except HPNE (Incell Corporation LLC, San Antonio, TX, U.S.A.) were purchased from Mediatech (Manassas, VA, U.S.A.) and were supplemented with 100 units/ml penicillin and 2 μg/ml streptomycin. All these cell lines have been routinely tested for mycoplasma contamination using the Universal Mycoplasma Detection Kit obtained from ATCC. Mycoplasma-free cell lines were used in all our experiments.

RNA isolation, reverse transcriptase PCR, and real-time PCR

RNA was isolated from cells using Trizol. The expression of the various genes was analyzed using either reverse transcriptase PCR or real-time PCR. After isolation, RNA concentration was measured using a Nanodrop ND-1000 system, followed by cDNA synthesis using the High Capacity cDNA Synthesis Kit (Invitrogen, Grand Island, NY, U.S.A.). Reverse transcriptase PCR was carried out under optimal conditions using a TaKaRa Taq Hot Start version (TaKaRa Bio USA Inc., Mountain View, CA, U.S.A.). The following primer pairs were used: human AhR, forward 5′-TCA AAT CCT TCC AAG CGG CA-3′ and reverse 5′-ACA GTT ATC CTG GCC TCC GT-3′; human ARNT, forward 5′-CCG GCA GAG AAT TTC AGG AAT AG-3′ and reverse 5′-GAA AGC TGC CCA CAC CAA AC-3′; human HPRT, forward 5′-GCG TCG TGA TTA GCG ATG ATG AAC-3′ and reverse 5′-CCT CCC ATC TCC TTC ATG ACA TCT-3′. In real-time PCR, the relative mRNA levels were measured with a SYBR Green detection system using the StepOnePlus real-time PCR system (Applied Biosystems, Foster City, CA, U.S.A.). The experiments were done in triplicates. The relative level of expression for each gene was calculated by normalizing the cycle threshold (Ct) value of the study gene to that of the housekeeping gene (hypoxanthine–guanine phosphoribosyltransferase-1 [HPRT1]). The following primer pairs were used: human CYP1A1, forward 5′-CAA GGG GCG TTG TGT CTT TG-3′ and reverse 5′-GTC GAT AGC ACC ATC AGG GG-3′; human CYP1A2 (cytochrome P450 family 1 subfamily A member 2), forward 5′-ATG AGA TGC TCA GCC TCG TG-3′ and reverse 5′-CCC GGA CAC TGT TCT TGT CA-3′; human CYP1B1 (cytochrome P450 family 1 subfamily B member 1), forward 5′-TCA CCA GGT ATC CTG ATG TGC-3′ and reverse 5′-CAG GAC ATA GGG CAG GTT GG-3′.

Immunofluorescence

Immunofluorescence studies were performed as described recently [20]. Briefly, cells grown on chamber slides were fixed, blocked, and stained with mouse anti-AhR antibody (Abcam, Cambridge, MA, U.S.A.) for 45 min after which they were washed and stained with secondary antibody (goat antimouse IgG) conjugated with Alexa Fluor 488 (Molecular Probes, Eugene, OR, U.S.A.), further washed and mounted with ProLong Diamond Antifade mountant with DAPI (4’,6-diamidino-2-phenyllindole, dihydrochloride), and the images were captured using a Nikon inverted microscope. Nuclei stained with DAPI are blue. Images were captured at 100× magnification.

Cell proliferation

Clonogenic assay was performed as described recently [20]. The cells were seeded at a very low density (500 cells/well) and allowed to attach to the substratum. They were then treated with different concentrations of Carbidopa (0, 1, 2.5, 5, 7.5, 10, 12.5, and 15 μM), and the colonies were allowed to grow for 2 weeks. The wells were washed every other day and fresh Carbidopa was added to the wells. At the end of the 2-week period, the wells were washed and the colonies were visualized with KaryoMax Giemsa stain after fixation with methanol. The stain was eluted with 1% sodium dodecyl sulfate in 0.2 N NaOH and the absorbance was measured at 630 nm.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation (ChIP) assay was performed using a ChIP Assay Kit (EMD Millipore Calbiochem, Bellerica, MA, U.S.A.) according to the manufacturer's protocol. The CYP1A1 promoter primers used were: (forward) 5′-AGC TAG GCC ATG CCA AAT-3′ and (reverse) 5′-AAG GGT CTA GGT CTG CGT GT-3′. HepG2 cells were seeded at 1.0 × 106 cells in a 10 cm dish. After 2 days, the cells were treated with Carbidopa (25 µM) or 3-methylcholanthrene (3-MC; 10 µM) for 6 h. DNA–protein complexes were cross-linked with 1% formaldehyde for 10 min at 37°C. After the cross-linking reaction was stopped with 1.25 M glycine, cells were washed with ice-cold PBS and harvested in ice-cold PBS containing protease inhibitors (Halt™ Protease and Phosphatase Inhibitor Cocktail, Thermo Fisher Scientific). Cells were centrifuged and lysed in the appropriate amount of lysis buffer (1.0 × 106 cells/200 µl) on ice for 10 min. Cell lysates were sonicated for six cycles (30 s ON, 60 s OFF) on ice to shear DNA. The sheared chromatin was centrifuged and the supernatant was diluted. Diluted supernatants were precleared with Protein A Agarose/Salmon sperm DNA (50% Slurry). After preclearing, diluted supernatants were incubated with 4 μg of AhR antibody (PRT9, Novus Biologicals, Littleton, CO, U.S.A.) overnight at 4°C. After the bound chromatin was washed, 5 M NaCl solution was added to 200 mM final concentration and the histone–DNA cross-links were reversed by incubation at 65°C for 4 h. The DNA–protein cross-links were reversed using proteinase K and the AhR-enriched fraction of genomic DNA was purified. PCR was used to analyze ChIP DNA for enrichment at a region on the CYP1A1 promoters. The DNA polymerase was heat-activated at 95°C for 4 min followed by 40 cycles, denaturing at 95°C for 45 s, annealing at 60°C for 45 s and elongating at 72°C for 45 s. PCR products were separated on 1.5% agarose gels and visualized by ethidium bromide staining.

Xenograft studies

Male athymic nu/nu mice (4 weeks old) were allowed to acclimatize to the environment for about a week before the start of the experiment. The animals were divided into control and treatment groups, and injected with phosphate-buffered saline (control group) and Carbidopa at 1 mg/mouse/day (treatment group) intraperitoneally, at least 5 days prior to tumor cell injection. BxPC-3 (10 × 106 cells in 30% matrigel) was injected subcutaneously at the right flank of the mice. The treatment continued all through the experimental period. Tumor size in different groups was measured using a caliper and the tumor volume was calculated using the formula (width2 × length)/2. Body weights of the animals were also recorded during the experimental phase. Following the animal sacrifice, the tumors were extracted and weighed from both the control and Carbidopa-treated mice.

Results and discussion

Inhibition of pancreatic cancer cell proliferation by Carbidopa and its potential relevance to the ability of the drug to inhibit indoleamine-2,3-dioxygenase 1

To test whether Carbidopa has anticancer effect, we used pancreatic cancer as a model. For this, we first conducted a colony formation assay with two human pancreatic cancer cell lines (BxPC-3 and Capan-2). Carbidopa (2.5 μM) significantly reduced the number of colonies in both the cell lines compared with the untreated controls (Figure 1A,B). This inhibitory effect was minimal in the normal pancreatic cell line hTERT-HPNE when treated with four times higher concentration of Carbidopa (10 μM) (Supplementary Figure S2), suggesting that the inhibition of colony formation by Carbidopa was almost selective for cancer cells. To corroborate these in vitro data, we performed subcutaneous xenografts in athymic nude mice using BxPC-3 cells. Carbidopa at a daily dose of 1 mg/mouse (intraperitoneal injection) significantly reduced the tumor volume compared with untreated controls (Figure 1C). A significant difference was also seen in terms of tumor weight between control and Carbidopa-treated mice (Figure 1D). Body weights between control and Carbidopa-treated mice remained unchanged (Figure 1E). These data provide evidence that Carbidopa possesses the ability to inhibit the proliferation of cancer cells in vitro and growth of tumors in nude mice in vivo.

Inhibition of pancreatic cancer cell proliferation by Carbidopa.

Figure 1.
Inhibition of pancreatic cancer cell proliferation by Carbidopa.

(A and B) Clonogenic assay in human pancreatic cancer cell lines (BxPC-3 and Capan-2) was cultured in the presence and absence of Carbidopa (1, 2.5, 5, 7.5, 10, 12.5, and 15 μM) for 2 weeks and stained with KaryoMax Giemsa stain. Data are given as means ± SEM. (C) Subcutaneous xenograft of BxPC-3 cells in athymic nude mice demonstrating difference in tumor volume between control and the Carbidopa-treated group. *P < 0.05 at all time points. (D) Tumor weights between BxPC-3/Control and BxPC-3/Carbidopa-treated mice. Data are given as means ± SEM. *P < 0.05. (E) Body weights between BxPC-3/Control and BxPC-3/Carbidopa-treated mice. Data are given as means ± SEM.

Figure 1.
Inhibition of pancreatic cancer cell proliferation by Carbidopa.

(A and B) Clonogenic assay in human pancreatic cancer cell lines (BxPC-3 and Capan-2) was cultured in the presence and absence of Carbidopa (1, 2.5, 5, 7.5, 10, 12.5, and 15 μM) for 2 weeks and stained with KaryoMax Giemsa stain. Data are given as means ± SEM. (C) Subcutaneous xenograft of BxPC-3 cells in athymic nude mice demonstrating difference in tumor volume between control and the Carbidopa-treated group. *P < 0.05 at all time points. (D) Tumor weights between BxPC-3/Control and BxPC-3/Carbidopa-treated mice. Data are given as means ± SEM. *P < 0.05. (E) Body weights between BxPC-3/Control and BxPC-3/Carbidopa-treated mice. Data are given as means ± SEM.

To date, the only known pharmacological action of Carbidopa is the inhibition of aromatic amino acid decarboxylase, but we are not aware of any direct association between this enzyme and cancer. Therefore, we looked for new, hitherto unknown, pharmacological actions for this drug that could be related to the observed anticancer effect. Carbidopa with its phenyl ring and a hydrazine moiety is structurally similar to phenylhydrazine, which is a potent inhibitor of indoleamine-2,3-dioxygenase 1 (IDO1) (IC50; 8 ± 2 µM); the inhibition occurs via interaction with heme at the active site of the enzyme [21]. IDO1 is a tryptophan-degrading enzyme [22] that is induced in antigen-presenting dendritic cells (DCs) in tumors and tumor-draining lymph nodes [2326]. The increased activity of IDO1 in DCs depletes the essential amino acid tryptophan in the surroundings, suppresses proliferation of cytotoxic T cells, and enables tumors to evade the immune system [2730]. Tumor cells themselves also up-regulate the expression of IDO1, suggesting potential involvement of the enzyme activity and also the resultant tryptophan metabolites as tumor promoters independent of the immune system [2830]. Currently, IDO1 inhibitors are in clinical trials as anticancer drugs [31,32]. The structural similarity between phenylhydrazine and Carbidopa suggested that the latter could also function as an IDO1 inhibitor. Although the anticancer effect of Carbidopa on pancreatic cancer cells was observed in our study in the absence of the immune system, it did not rule out the potential role of IDO1 inhibition by Carbidopa in tumor cells themselves. Therefore, we first employed a computer-based molecular docking modeling using the programs AutoDock4 and Gold 5.0, which suggested that Carbidopa could function as an IDO1 inhibitor (data not shown). To test the validity of the computer prediction, we studied the effects of Carbidopa on human recombinant IDO1 by measuring the activity of the enzyme using a colorimetric assay. This experiment confirmed that Carbidopa does indeed inhibit IDO1 (Supplementary Figure S3). During the assay, we discovered that the colorimetric assay was not suitable to determine accurately the efficacy of Carbidopa as an IDO1 inhibitor. The assay measures the product of the enzymatic activity, namely kynurenine, with the formation of a yellow-colored product, but Carbidopa itself gets oxidized while standing at room temperature to yield a yellow-colored product. As a result, the efficacy of Carbidopa for IDO1 inhibition deduced from the colorimetric assay was markedly underestimated. Nonetheless, the data indicate that the anticancer effect of Carbidopa on pancreatic cancer could be due to its IDO1 inhibition because pancreatic tumors and cancer cells express IDO1 (data not shown).

Carbidopa is a high-affinity agonist for the nuclear receptor AhR

As we realized the unsuitability of the colorimetric assay to accurately determine the potency of Carbidopa for the inhibition of IDO1, we used an alternative approach. IDO1 degrades tryptophan, and kynurenine is the product of this activity. Kynurenine is a known physiological agonist for the nuclear receptor AhR [33]. Agonist-induced activation of AhR is widely monitored by the induction of the AhR target gene CYP1A1 [34]. Therefore, we decided to assess the efficacy of Carbidopa as an IDO1 inhibitor by monitoring CYP1A1 expression in intact cells. Our rationale was simple (Figure 2A). If Carbidopa inhibits IDO1 in an intact cell, kynurenine generation would be decreased, thus resulting in the suppression of AhR transcriptional activity, which can be monitored from decreased expression of CYP1A1 in Carbidopa-treated cells. Unliganded AhR resides in the cytoplasm, forming a complex with heat shock proteins. Upon binding an agonist, the cytoplasmic complex dissociates and the ligand-bound AhR gets translocated to the nucleus; AhR nuclear translocator (ARNT) facilitates the dissociation of the complex in the cytoplasm as well as the translocation of the ligand-bound AhR to the nucleus. The AhR–ARNT dimer then binds to a dioxin-responsive element in target genes and induces transcription. CYP1A1 is one of the target genes [34]. Therefore, it should be possible to monitor the inhibition of IDO1 by Carbidopa by determining the efficacy of the drug on CYP1A1 expression; this approach would circumvent the drawback that we encountered in the colorimetric assay. With this rationale, we first checked the mRNA expression of AhR and its binding partner ARNT in BxPC-3 and Capan-2 cells (Figure 2B). These genes were expressed in both pancreatic cancer cell lines. We then treated these cancer cells with Carbidopa for 6 h and examined the expression levels of CYP1A1 mRNA. Surprisingly, these experiments produced results that were opposite to our original rationale. Carbidopa did not decrease CYP1A1 expression as expected; it increased the expression of this AhR target gene in a dose-dependent manner in BxPC-3 cells (Figure 2C) as well as in Capan-2 cells (Figure 3A). These data suggested that Carbidopa could actually be an agonist for AhR. To confirm that the Carbidopa-induced up-regulation of CYP1A1 indeed occurred via AhR activation, we used CH223191, an AhR-specific antagonist. The ability of Carbidopa to induce CYP1A1 was blocked by this antagonist in both BxPC-3 cells (Figure 2D) and Capan-2 cells (Figure 3B), clearly demonstrating the involvement of AhR activation in Carbidopa-induced expression of CYP1A1. To further corroborate our data, we used resveratrol, another AhR antagonist; similar results were obtained in both cell lines (Figure 3C,D). Ligand-dependent activation of AhR is associated with the translocation of the receptor from the cytoplasm into nucleus. Therefore, we used immunofluorescence to localize AhR in control and Carbidopa-treated BxPC-3 and Capan-2 cells. These studies provided evidence of nuclear translocation of the receptor in Carbidopa-treated cells (Figure 2E,F). These data provide strong evidence that Carbidopa is an agonist for AhR. This was a serendipitous discovery during the course of our efforts to characterize Carbidopa as an inhibitor of IDO1.

Carbidopa induces CYP1A1 expression in an AhR-dependent manner in pancreatic cancer cells.

Figure 2.
Carbidopa induces CYP1A1 expression in an AhR-dependent manner in pancreatic cancer cells.

(A) Rationale for using AhR activation and its downstream target gene CYP1A1 expression as a readout for the inhibition of IDO1 by Carbidopa. (B) RT-PCR demonstrating the expression of AhR and ARNT in pancreatic cancer cell lines (BxPC-3 and Capan-2). HPRT was taken as an internal control. (C) Real-time RT-PCR demonstrating the increase in CYP1A1 expression in BxPC-3 cells following 6-h treatment with varying concentrations of Carbidopa. Columns, relative to control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05 compared with untreated control. (D) Real-time RT-PCR showing the relative CYP1A1 expression in BxPC-3 cells following 6-h treatment with Carbidopa (10 μM) in the presence or absence of the AhR antagonist CH223191 (10 μM). Columns, relative to control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05. (E and F) Immunofluorescence detection of AhR (green) in control and Carbidopa-treated (25 μM for 6 h) BxPC-3 and Capan-2 cells. Nuclei stained with DAPI are blue. Magnification, 100×.

Figure 2.
Carbidopa induces CYP1A1 expression in an AhR-dependent manner in pancreatic cancer cells.

(A) Rationale for using AhR activation and its downstream target gene CYP1A1 expression as a readout for the inhibition of IDO1 by Carbidopa. (B) RT-PCR demonstrating the expression of AhR and ARNT in pancreatic cancer cell lines (BxPC-3 and Capan-2). HPRT was taken as an internal control. (C) Real-time RT-PCR demonstrating the increase in CYP1A1 expression in BxPC-3 cells following 6-h treatment with varying concentrations of Carbidopa. Columns, relative to control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05 compared with untreated control. (D) Real-time RT-PCR showing the relative CYP1A1 expression in BxPC-3 cells following 6-h treatment with Carbidopa (10 μM) in the presence or absence of the AhR antagonist CH223191 (10 μM). Columns, relative to control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05. (E and F) Immunofluorescence detection of AhR (green) in control and Carbidopa-treated (25 μM for 6 h) BxPC-3 and Capan-2 cells. Nuclei stained with DAPI are blue. Magnification, 100×.

Activation of AhR by Carbidopa in the human pancreatic cancer cell lines Capan-2 and BxPC-3.

Figure 3.
Activation of AhR by Carbidopa in the human pancreatic cancer cell lines Capan-2 and BxPC-3.

(A) Relative CYP1A1 mRNA expression in Capan-2 cells cultured in the absence and presence of varying concentrations of Carbidopa for 6 h. (B and C) Real-time RT-PCR showing inhibition of Carbidopa-induced CYP1A1 expression following treatment with AhR antagonists CH223191 and resveratrol in Capan-2 cells. (D) Real-time RT-PCR showing inhibition of Carbidopa-induced CYP1A1 expression following treatment with resveratrol in BxPC-3 cells. Columns, relative to control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05 compared with untreated control.

Figure 3.
Activation of AhR by Carbidopa in the human pancreatic cancer cell lines Capan-2 and BxPC-3.

(A) Relative CYP1A1 mRNA expression in Capan-2 cells cultured in the absence and presence of varying concentrations of Carbidopa for 6 h. (B and C) Real-time RT-PCR showing inhibition of Carbidopa-induced CYP1A1 expression following treatment with AhR antagonists CH223191 and resveratrol in Capan-2 cells. (D) Real-time RT-PCR showing inhibition of Carbidopa-induced CYP1A1 expression following treatment with resveratrol in BxPC-3 cells. Columns, relative to control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05 compared with untreated control.

Liver is the major tissue where AhR activation plays a key role in xenobiotic metabolism. Therefore, we decided to confirm our findings that Carbidopa is an AhR agonist using the human liver cell line HepG2. As expected, Carbidopa treatment led to CYP1A1 induction as early as 3 h post-treatment (Figure 4A); the induction was dose-dependent (Figure 4B). Under similar conditions, l-DOPA did not affect CYP1A1 expression. Furthermore, as observed in pancreatic cancer cells, AhR-specific antagonists CH223191 and resveratrol blocked Carbidopa-induced CYP1A1 expression (Figure 4C,D). Carbidopa treatment also led to the nuclear translocation of AhR (Figure 4E).

Carbidopa induces CYP1A1 expression in an AhR-dependent manner in HepG2, a human liver cancer cell line.

Figure 4.
Carbidopa induces CYP1A1 expression in an AhR-dependent manner in HepG2, a human liver cancer cell line.

(A) Relative CYP1A1 mRNA expression following the treatment of HepG2 cells with 10 μM Carbidopa for various time intervals. Data points are relative to untreated control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05 compared with untreated control, which was taken as 1. (B) Relative CYP1A1 mRNA expression in HepG2 cells cultured in the absence and presence of varying concentrations of Carbidopa and 100 μM l-DOPA for 6 h. Columns, relative to untreated control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05 compared with untreated control. (C and D) Relative CYP1A1 mRNA expression in HepG2 cells treated with Carbidopa (10 μM) in the presence or absence of two different AhR antagonists: CH223191 (10 μM) and resveratrol (20 μM) for 6 h. Columns, relative to control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05. (E) Immunofluorescence detection of AhR (green) in control and Carbidopa-treated (25 μM for 6 h) HepG2 cells. Nuclei stained with DAPI are blue. Magnification, 100×.

Figure 4.
Carbidopa induces CYP1A1 expression in an AhR-dependent manner in HepG2, a human liver cancer cell line.

(A) Relative CYP1A1 mRNA expression following the treatment of HepG2 cells with 10 μM Carbidopa for various time intervals. Data points are relative to untreated control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05 compared with untreated control, which was taken as 1. (B) Relative CYP1A1 mRNA expression in HepG2 cells cultured in the absence and presence of varying concentrations of Carbidopa and 100 μM l-DOPA for 6 h. Columns, relative to untreated control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05 compared with untreated control. (C and D) Relative CYP1A1 mRNA expression in HepG2 cells treated with Carbidopa (10 μM) in the presence or absence of two different AhR antagonists: CH223191 (10 μM) and resveratrol (20 μM) for 6 h. Columns, relative to control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05. (E) Immunofluorescence detection of AhR (green) in control and Carbidopa-treated (25 μM for 6 h) HepG2 cells. Nuclei stained with DAPI are blue. Magnification, 100×.

With a fixed concentration (10 μM), we compared in HepG2 cells the induction of CYP1A1 by Carbidopa with that by other known AhR agonists, which included 3-MC, benzo[a]pyrene, kynurenine, indole-3-carbinol, and indole acetic acid (Table 1). The efficacy of Carbidopa to activate the receptor is in the same range as that of kynurenine, one of the known physiological agonists for AhR.

Table 1
Effects of Carbidopa and known AhR agonists (10 μM) on CYP1A1 expression in HepG2 cells
Compound Fold (range) 
Carbidopa 2.63 (2.45–2.82) 
3-Methylcholanthrene 56.8 (54.7–59.0) 
Benzo[a]pyrene 35.3 (32.4–38.5) 
Kynurenine 3.97 (3.82–4.14) 
Indole-3-carbinol 7.62 (7.37–7.88) 
Indole acetic acid 14.2 (14.0–14.4) 
Compound Fold (range) 
Carbidopa 2.63 (2.45–2.82) 
3-Methylcholanthrene 56.8 (54.7–59.0) 
Benzo[a]pyrene 35.3 (32.4–38.5) 
Kynurenine 3.97 (3.82–4.14) 
Indole-3-carbinol 7.62 (7.37–7.88) 
Indole acetic acid 14.2 (14.0–14.4) 

To further characterize Carbidopa as an AhR agonist, we employed ChIP assay for confirmation. AhR is a transcription factor and, upon activation by its ligand, gets translocated to the nucleus as a dimer with ARNT. There it binds to the xenobiotic (dioxin)-responsive element in its target genes and induces their expression. The ChIP assay showed unequivocally that Carbidopa (25 µM) induced AhR binding to the promoter of CYP1A1, one of the target genes for AhR. Similar ChIP results were obtained with 3-MC, a known AhR agonist. The binding of AhR to the CYP1A1 promoter did not occur in untreated control cells (Figure 5A). To further corroborate the promotion of AhR signaling by Carbidopa, we checked the expression of other AhR target genes (CYP1A2 and CYP1B1). The mRNA expression of CYP1A2 and CYP1B1 significantly increased in BxPC-3 (Figure 5B,C), Capan-2 (Figure 5D,E), and HepG2 (Figure 5G,H) cell lines in a dose-dependent manner following 6-h treatment with Carbidopa (10 and 50 µM), further supporting the fact that Carbidopa is indeed an AhR agonist.

Activation of cytochrome P450 enzymes by Carbidopa is AhR-dependent.

Figure 5.
Activation of cytochrome P450 enzymes by Carbidopa is AhR-dependent.

(A) ChIP assay of AhR binding to the CYP1A1 promoter. HepG2 cells were treated with Carbidopa (25 µM) or 3-MC (10 µM) for 6 h. AhR binding to the CYP1A1 gene promoter was determined in a ChIP assay as described in Materials and Methods. (B and C) Relative CYP1A2 and CYP1B1 mRNA expression in BxPC-3 cells cultured in the absence and presence of Carbidopa (10 and 50 μM) for 6 h. (D and E) Relative CYP1A2 and CYP1B1 mRNA expression in Capan-2 cells cultured in the absence and presence of Carbidopa (10 and 50 μM) for 6 h. (F and G) Relative CYP1A2 and CYP1B1 mRNA expression in HepG2 cells cultured in the absence and presence of Carbidopa (10 and 50 μM) for 6 h. Columns, relative to untreated control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05 compared with untreated control.

Figure 5.
Activation of cytochrome P450 enzymes by Carbidopa is AhR-dependent.

(A) ChIP assay of AhR binding to the CYP1A1 promoter. HepG2 cells were treated with Carbidopa (25 µM) or 3-MC (10 µM) for 6 h. AhR binding to the CYP1A1 gene promoter was determined in a ChIP assay as described in Materials and Methods. (B and C) Relative CYP1A2 and CYP1B1 mRNA expression in BxPC-3 cells cultured in the absence and presence of Carbidopa (10 and 50 μM) for 6 h. (D and E) Relative CYP1A2 and CYP1B1 mRNA expression in Capan-2 cells cultured in the absence and presence of Carbidopa (10 and 50 μM) for 6 h. (F and G) Relative CYP1A2 and CYP1B1 mRNA expression in HepG2 cells cultured in the absence and presence of Carbidopa (10 and 50 μM) for 6 h. Columns, relative to untreated control. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). *P < 0.05 compared with untreated control.

Up-regulation of AhR in pancreatic cancer is suggestive of its role as a drug target to treat pancreatic cancer

The experiments described thus far have uncovered AhR as a novel pharmacological target for Carbidopa; this drug is an agonist for AhR. To assess if this new molecular target for Carbidopa has any relevance to pancreatic cancer, we examined the expression of AhR in pancreatic cancer cell lines, PDXs (patient-derived xenografts), and primary pancreatic tumor tissues. AhR expression was up-regulated in majority of the PDXs examined compared with normal pancreatic tissues (Figure 6A). Pancreatic cancer cell lines also showed up-regulation of AhR compared with normal cell lines (hTERT-HPNE and HPDE) (Figure 6B). The same was also true with primary tumor samples (data not shown). We also analyzed the publicly available microarray datasets for pancreatic cancer and found AhR up-regulation in cancer (Figure 6C), clearly suggesting that AhR could serve as a drug target to treat pancreatic cancer.

Increased AhR expression in pancreatic cancer.

Figure 6.
Increased AhR expression in pancreatic cancer.

(A) Real-time RT-PCR showing relative AhR expression in pancreatic normal tissues vs. PDXs of pancreatic cancer. Columns, relative to normal. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). (B) RT-PCR showing the expression of AhR in pancreatic normal (hTERT-HPNE and HPDE) and cancer cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, MIA PaCa-2, and Panc-1). HPRT was taken as an internal control. (C) Box plots for the expression of AhR in pancreatic tumor tissues compared with pancreatic normal tissue, as assessed from the publicly available microarray datasets. The horizontal line within each box represents the median value. The box edges represent the lower (25th) and upper (75th) quartile.

Figure 6.
Increased AhR expression in pancreatic cancer.

(A) Real-time RT-PCR showing relative AhR expression in pancreatic normal tissues vs. PDXs of pancreatic cancer. Columns, relative to normal. Error bars indicate 95% confidence interval estimates of the mean expressions (n = 3). (B) RT-PCR showing the expression of AhR in pancreatic normal (hTERT-HPNE and HPDE) and cancer cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2, MIA PaCa-2, and Panc-1). HPRT was taken as an internal control. (C) Box plots for the expression of AhR in pancreatic tumor tissues compared with pancreatic normal tissue, as assessed from the publicly available microarray datasets. The horizontal line within each box represents the median value. The box edges represent the lower (25th) and upper (75th) quartile.

To summarize, our study has identified Carbidopa as an AhR agonist. The dose–response data show that Carbidopa activates AhR at concentrations of 3–10 μM; these concentrations are therapeutically relevant. The recommended dose for Carbidopa in humans for the treatment of PD is 200 mg/day, but the drug is safe even at a dose as high as 450 mg/day [35]. With these doses of oral administration, plasma concentrations of the drug can reach levels sufficient to activate AhR (2 μM at 75 mg/day dose; 16 μM at 450 mg/day dose) [35]. The findings of the present study that Carbidopa at a dose of 1 mg/mouse/day is effective in blocking pancreatic tumor growth in vivo are also therapeutically relevant. This effective dose in mice translates to a human dose of <400 mg/day (allometric scaling calculations from the FDA Draft guidelines) [36,37]. Currently, the prevailing notion is that AhR and its ability to induce selective cytochrome P450 enzymes are primarily related to xenobiotic detoxification and to the efficacy of drugs, including anticancer drugs [38]. But, recent studies have uncovered a critical role for AhR in cancer [39] and that AhR activation is effective to treat many types of cancer including breast cancer, colon cancer, and pancreatic cancer [13,14,4042]. Our findings that Carbidopa is an AhR agonist suggest that this drug has potential for cancer treatment. That said, the role of AhR in carcinogenesis is complex and ligands may either promote or inhibit carcinogenesis in a tumor-specific manner [13]. As Carbidopa is already used to treat PD, further investigations to potentially re-purpose the drug for cancer treatment are clearly warranted.

Abbreviations

     
  • 3-MC

    3-methylcholanthrene

  •  
  • AhR

    aryl hydrocarbon receptor

  •  
  • ARNT

    AhR nuclear translocator

  •  
  • ChIP

    chromatin immunoprecipitation

  •  
  • CYP1A1

    cytochrome P450 family 1 subfamily A member 1

  •  
  • CYP1A2

    cytochrome P450 family 1 subfamily A member 2

  •  
  • CYP1B1

    cytochrome P450 family 1 subfamily B member 1

  •  
  • DAPI

    4’,6-diamidino-2-phenyllindole, dihydrochloride

  •  
  • DMEM

    Dulbecco's Modified Eagle's Medium

  •  
  • FBS

    fetal bovine serum

  •  
  • HPDE

    a human pancreatic ductal epithelial cell line

  •  
  • HPRT1

    hypoxanthine–guanine phosphoribosyltransferase-1

  •  
  • IDO1

    indoleamine 2,3-dioxygenase 1

  •  
  • l-DOPA

    l-3,4-dihydroxyphenylalanine

  •  
  • PD

    Parkinson's disease

  •  
  • PDX

    patient-derived xenograft

Author Contribution

J.O. performed most of the experiments; S.M. and K.S. did the computer modeling studies; S.Y. was responsible for analyzing the mRNA expression of AhR in pancreatic tumor tissues from online databases; S.G. performed the IDO1 assay; V.G. contributed to the design of the study and in the preparation of the manuscript; Y.D.B. was responsible for the design of the study, interpretation of the data, and writing of the manuscript.

Funding

This work was supported by grants from the South Plains Foundation and the CH Foundation, Texas, and also by start-up funds from the Texas Tech University Health Sciences Center.

Competing Interests

The Authors declare that there are no competing interests associated with the manuscript.

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Supplementary data