Gold (I) N-heterocyclic carbene complex inhibits mouse melanoma growth by p53 upregulation

The cytotoxic effect of complex 3 was investigated on HCT 116, HepG2, A549 and B16F10 cells by MTT assay. Treatment of different concentrations of complex 3 (0 to 50 μM) reduced the viability of the cancer cells in a dose dependent manner (Figure 2A) in different degrees, with higher toxicity in HCT 116 (GI₅₀ 4.73 ± 0.20 μM) after 24 h. It also showed good cytotoxic activity on the other three cell lines, HepG2 (GI₅₀ 9.48 ± 0.25 μM), B16F10 (GI₅₀ 9.4 ± 0.26 μM) and A549 (GI₅₀ 13.71 ± 0.15 μM) after 24 h. In a time dependent study, complex 3 (GI₅₀ concentrations) induced 77% and 75% mortality in HCT 116 and HepG2 cells respectively, 67% mortality in B16F10 cells and 60% mortality in A549 cells after 48 h (Figure 2B and Additional file 6: Table S2). The commonly used anticancer drug cisplatin had a marginally higher rate of cytotoxicity as compared to 3 after 24 h (Figure 2C and Additional file 6: Table S1). However, the proligand 2 failed to elicit any cytotoxicity in the cancer cell lines mentioned in this study. Thus, complex 3 is a good cytotoxic agent against the cancer cells used in this study.

B16F10 is a highly metastatic form of a mouse melanoma cell line [17]. Complex 3 showed significant cytotoxicity towards this cell line like the other cancer cell lines used in this study. Therefore B16F10 cells were used as model for further study on cytotoxicity induced by complex 3. Cancer cell death through apoptosis is a desirable goal. Complex 3 showed characteristic apoptotic changes in morphology like cell shrinkage and rounding (Figure 2D, upper panel), and nuclear changes such as chromatin condensation and DNA fragmentation, seen using the nuclear staining dye AO along with EtBr and DAPI (Figure 2D, middle panel and lower panel respectively). The number of condensed bright fragmented nuclei seen in complex 3 treated cells after DAPI staining was higher, while yellowish, orange and red colored nuclei or a combination of these colored nuclei were observed upon staining with AO/EtBr. Marked increase in the number of DNA fragments in complex 3 treated cells as seen by using the DNA fragmentation assay kit (Figure 2G) also corroborated the findings in Figure 2D. Thus, cytotoxicity induced by complex 3 in B16F10 cells may involve apoptosis.

Phosphatidylserine [PS] externalization from inner cell membrane to outer membrane is a prerequisite for apoptosis. Externalized PS can bind with annexin V [21]. B16F10 cells exposed to complex 3 showed higher percentage of annexin V-FITC binding cells after staining with annexin V-FITC and PI. After 24 h of treatment with 10 μM of complex 3, percentage of apoptotic cells was 79.2% as compared to 3.9% in the vehicle treated cells (Figure 2E). These findings suggested that complex 3 induced death of B16F10 cells resulting from apoptosis.

Cell cycle arrest at various phases of cell division precedes apoptosis [13]. There was a gradual decrease in the number of cells in G1 phase upon treatment with complex 3 (0 and 10 μM) for 24 h (Figure 2F). The percentage of G0–G1 population for treated cells was 68.7% at 24 h as compared to 73.9% for vehicle treated cells, indicating a decrease of 5.2% of cell population in the G0–G1 phase. The percentages of S phase population for such cells treated with complex 3 (0 and 10 μM) were 13.0% and 15.8% respectively indicating an increase in 2.8% of cell population in the S phase after 24 h. Moreover, the percentages of G2-M phase population for complex 3 (0 and 10 μM) treated B16F10 cells at 24 h were 13.0% and 15.4% respectively indicating an increase of 2.4% of cell population in the G2-M phase. This indicated that complex 3 may mediate cell cycle arrest at the G2-M phase.

“Apoptotic trigger” is initiated when the ratio of the pro- and anti-apoptotic members of the Bcl-2 family is unbalanced. Downregulation of the anti-apoptotic Bcl-2 protein and binding of pro-apoptotic Bax protein to the mitochondria membrane cause disruption of the mitochondrial membrane leading to the release of cytochrome c from mitochondria to cytosol. These events lead to the activation of caspase 9 (initiator caspase) and caspase 3 (executioner caspase). Activated caspases induce the cleavage of PARP, thereby perturbing its ability for DNA repair and leading to cell death [13]. Treatment of complex 3 suppressed the level of Bcl-2, activated Bax, lowered the expressions of pro-caspase-3 and pro-caspase-9, promoted PARP cleavage, and increased the level of cytosolic cytochrome c in a dose dependent manner (Figure 3A). Densitometric analyses of the western blots have been shown in Figure 3B. Status of active caspase-3 and caspase-9, which increased with doses of complex 3 as seen in the study using assay kit (Figure 3C), were dampened upon pre-incubation of cells with Z-LEHD-FMK and Z-DEVD-FMK (Figure 3D). Moreover cells pre-incubated with Z-LEHD-FMK and Z-DEVD-FMK exhibited higher cell viability as compared to cells treated with either one or none of these inhibitors prior to treatment with complex 3 (Figure 3E). Elevated level of cytosolic cytochrome c was also observed (Figure 3F) 24 h after complex 3 treatment (0 and 10 μM), along with a dose dependent decrease in ∆Ψm (Figure 3G). Moreover, flowcytometric analysis of vehicle treated cells revealed that 99.5% of the cell population exhibited fluorescence at the PE-Texas Red A channel indicating a higher level of cells having a healthy ∆Ψm, whereas complex 3 (10 μM) treated cells revealed that 52.0% of the cell population fluorescence at the PE-Texas Red A channel, hinting at a loss of ∆Ψm in 47.5% of cell population after 24 h (Figure 3H). These results indicate that complex 3 may induce apoptosis via the mitochondrial pathway.

Generation of reactive oxygen species (ROS) is a key factor of apoptotic cell death. The maximum ROS generation was exhibited at 12 h after treatment of cells with complex 3 (Figure 3I). Treatment with NAC (N-acetyl-cysteine) 1 h prior to complex 3 treatment resulted in decrease in ROS production (Figure 3J) along with inhibition of cell death (Figure 3K). Also, flowcytometric analysis revealed that the FITC mean intensity was 410 in vehicle treated cells but 8829 in complex 3 (10 μM) treated cells after 24 h, indicating a shift in FITC mean intensity from the vehicle treated cells to the cells treated with complex 3 (10 μM) (Figure 3L). However, incubation with NAC 1 h prior to complex 3 (0 and 10 μM) treatment resulted in a mean FITC mean intensity of 956 for vehicle treated cells but 1928 for 3 (10 μM) treated cells after 24 h, indicating a shift in intensity from the vehicle treated cells to the cells treated with complex 3 (10 μM) (Figure 3L). The study demonstrates that complex 3 induced apoptosis in B16F10 may proceed via the ROS mediated pathway.

Apoptosis is generally regulated by p53. Therefore, the role of complex 3 on p53 activation was examined. Treatment of B16F10 cells with complex 3 (0, 5 and 10 μM) resulted in upregulation of p53, p21 and phospho-p53 (p-p53) (Figure 4A). Treatment with 10 μM of complex 3 for 24 h resulted in increased translocation of p53 to the nucleus along with increased expression of p21 as compared to the untreated cells (Figure 4B). The corresponding fluorescence intensity graphs have been provided in a supplementary figure (Additional file 7: Figure S6). Pretreatment of pifthrin-α (PFT-α), an inhibitor of p53 transactivation, inhibited the expression of p21 along with reduction in the level of p53 dependent proteins such as Bax and cytosolic cytochrome c. However, PFT-α failed to downregulate the expression of p-p53 (ser 15) whereas p53 registered a slight reduction in its expression (Figure 4C-D). Also, pretreatment with PFT-α prevented growth inhibition of cells treated with complex 3 (Figure 4E). Therefore p53 may play a significant role in complex 3 mediated apoptosis of B16F10 cells.

Treatment of B16F10 cells with NAC 1 h prior to complex 3 addition led to the inhibition of p53 as well as p-p53 (phosphorylated at ser-15) (Figure 4F). However, treatment of PFT-α failed to suppress ROS production (Figure 4G), indicating that p53 may not be involved in ROS generation whereas ROS maybe involved in p-p53 up regulation.

Complex 3 administered intraperitoneally at the dosages of 5, 10, 20, and 40 mg/kg body weight per day till the 60^th day showed 100% survivability for dosages of 0, 5 and 10 mg/kg, 80% survivability with 20 mg/kg, and 70% survivability with 40 mg/kg body weight respectively (Figure 5A). Administration of complex 3 (0, 5 and 10 mg/kg body weight/day) was started from 15^th day onward since the day of injection of B16F10 cells in the mice and was continued till the 23^rd day. The mice were sacrificed on the 24^th day (Figure 5B).

Mice bearing the B16F10 tumor but not administered complex 3 faced total mortality by the 40^th day, whereas treatment with 5 and 10 mg/kg mouse body weight of the complex ensured 30% and 70% survivability till the 60^th day (Figure 5C). Dose dependent decrease in the tumor size was observed following treatment with complex 3 (0, 5 and 10 mg/kg of mouse body weight) (Figure 5D). Haematoxylin and Eosin staining revealed an increase in number of fragmented nuclei in a dose dependent manner (Figure 5G). Both mitotic index (an indication of cell proliferation) and tumor weight decreased upon treatment with complex 3 (Figure 5H-I). Body weight remained nearly constant (Figure 5E), but the tumor volume decreased in a dose dependent manner (Figure 5F). The results show that complex 3 is a potent suppressor of melanoma tumor.

The p53 protein expression and translocation to the nucleus was upregulated along with the upregulation of p21 in the tumor sections of the mice administered with complex 3 (10 mg/kg body weight of mice) with respect to the tumors sections of the mice not administered complex 3 (Figure 6, upper panel). The translocation of the anti-apoptotic NF-κB protein (p65 and p50) was inhibited upon administration of complex 3 (10 mg/kg body weight of mice) (Figure 6, middle panel). Following treatment with complex 3 (10 mg/kg body weight of mice), the expressions of angiogenic and metastatic markers such as VEGF and MMP-9 respectively were also inhibited (Figure 6, lower panel), indicating that tumor death occurs by activation of p53 and inhibition of the NF-κB protein, VEGF and MMP-9. The corresponding fluorescence intensity graphs have been provided as a supplementary figure (Additional file 8: Figure S7).

Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin, neomycin (PSN) antibiotic, trypsin and ethylene di-amine tetra-acetic acid (EDTA) were obtained from Gibco BRL (Grand Island, NY, USA). Tissue culture plastic wares were obtained from NUNC (Roskilde, Denmark). Antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Organic solvents used were of HPLC grade. All other chemicals including doxorubicin, NAC used were from Sigma Chem. Co. (St. Louis, MO, USA) or mentioned otherwise.

Pyridine-2-carboxaldehyde (450 mg, 4.21 mmol) and N-acetylethylenediamine (429 mg, 4.21 mmol) were taken in dry ethanol (25 ml) and refluxed under stirring condition for 6 h. Volatiles were removed under reduced pressure and dried under vacuum. Analytically pure samples were obtained after crystallization by slow diffusion of diethyl ether into chloroform solution of Schiff base. The solid mass was dried in vacuum and the yield was 68% (547 mg, 2.86 mmol).

2-Pyridyl-N-(2-ethylacetoamido) methylamine 1 (450 mg, 2.36 mmol) and crushed 91% paraformaldehyde powder (75.6 mg, 2.70 mmol) were taken in 20 ml of dioxane and stirred for 4 h to form a slurry; then the mixture was refluxed for 10 h. A 5 ml aliquot of 2 (N) HCl in diethyl ether was added slowly to the cold slurry, resulting in immediate separation of layers - a lower layer of a yellowish red viscous liquid and a colorless liquid in the upper part. Then 5 ml water was added to the solution and the aqueous phase was separated by a separating funnel. Excess saturated aqueous KPF₆ was added to the solution and immediately a white precipitate was obtained. This precipitate was then filtered out and recrystallized from acetonitrile and diethyl ether. The resultant solid mass was dried in vacuum and the yield was 66% (542 mg, 1.55 mmol).

Proligand 2 (200 mg, 0.57 mmol) and silver oxide (67.2 mg, 0.29 mmol) were dissolved in acetonitrile (15 ml) and the mixture was stirred for 5 h. The volume of the solution containing a silver carbene complex was reduced to 5 ml. In 5 ml acetonitrile solution of AuSMe₂Cl (85.4 mg, 0.29 mmol), the silver carbene complex was added drop wise, and immediately a white precipitate was observed. The mixture was filtered; the volatiles were removed under reduced pressure and dried under vacuum. Analytically pure sample of a gold (I) carbene complex 3 was obtained after crystallization by slow diffusion of diethyl ether into an acetonitrile solution of complex 3. Yield was 80% (173.5 mg, 0.23 mmol).

Cell lines, such as HCT-116 (human colorectal carcinoma), HepG2 (human hepatocellular carcinoma), A549 (human non small lung carcinoma) and B16F10 (mouse melanoma), were obtained from National Centre for Cell Science, Pune, India. These cell lines were cultured in DMEM supplemented with 10% FBS and 1% antibiotic (PSN) and incubated at 37°C in a humidified atmosphere with 5% CO₂. After achieving 75–80% confluence, cells were harvested with 0.025% trypsin and 0.52 mM EDTA in phosphate buffered saline (PBS) and were seeded at desired density to allow them to re-equilibrate a day before the start of experimentation. All experiments were conducted in DMEM supplemented with 10% FBS and 1% antibiotic (PSN) solution.

Cells were treated with various concentrations of complex 3 (0, 2.5, 5, 10, 25 and 50 μM) dissolved in 0.05% of DMSO for 24 h and their respective GI₅₀s were determined. Moreover, viability of cells treated with the GI₅₀ concentrations of complex 3 (determined after 24 h) for 12, 24, 36 and 48 h were determined by MTT assay as described earlier [28]. In another set of experiments, cells treated with cisplatin (0, 2.5, 5, 10, 25 and 50 μM) for 24 h were taken as positive control. Absorbance of the solubilized intracellular formazan was measured at 595 nm using an ELISA reader (Model: Emax, Molecular device, USA).

Apoptosis was assayed by using an annexin V-FITC apoptosis detection kit (Calbiochem, La Jolla, CA) as described earlier [21]. B16F10 cells treated with complex 3 (0 and 10 μM) for 24 h were stained with PI and annexin V-FITC according to manufacturer’s instructions. The percentage of live, apoptotic and necrotic cells were analyzed by BD LSRFortessa cell analyzer (Becton Dickinson, San Jose, CA, USA). Data from 10⁶ cells were analyzed for each sample.

B16F10 cells were treated with complex 3 (0, 2.5, 5, 10 and 25 μM) for 24 h and fragmented DNA was assayed by an ELISA based method as earlier described [13].

Cell cycle arrest was analyzed by treating cells with complex 3 (0 and 10 μM) for 24 h followed by PI staining, as described earlier [13]. The percentages of cell population undergoing cell cycle arrest at various stages of mitosis were analyzed by BD LSRFortessa cell analyzer (Becton Dickinson, San Jose, CA, USA). Data from 10⁶ cells were analyzed for each sample.

B16F10 cells were treated with complex 3 (0, 2.5, 5, 10 and 25 μM) for 24 h and caspase-3 and caspase-9 activities were quantified with commercially available caspase-3/CPP32 and caspase-9 colorimetric Assay kit (BioVision Research Products, Mountain View, CA) as described earlier [13]. Caspase activity was spectrophotometrically detected at 405 nm on an ELISA reader (Model: Emax, Molecular device, USA). In another set of experiments, B16F10 cells were treated with 10 μM of Z-DEVD-FMK (caspase-3 inhibitor) and Z-LEHD-FMK (caspase-9 inhibitor) 1 h prior to treatment with complex 3 (0 and 10 μM) and MTT assay was done to evaluate growth inhibition of cells.

For the detection of intracellular ROS generation, B16F10 cells treated with complex 3 (0 and 10 μM) for 24 h were incubated with 10 μM of H2DCFH-DA (2′, 7′-dichlorofluorescein diacetate, Molecular Probes) with or without NAC (N-acetyl-cysteine, added 1 h prior to complex 3 treatment) for 25 min at 37˚C, following which cells were analyzed by BD LSRFortessa cell analyzer. Data from 10⁶ cells were analyzed for each sample as described earlier [13]. In another set of experiments, B16F10 cells treated with complex 3 (0, 2.5, 5, 10 and 25 μM) for 6, 12 and 24 h were incubated with 10 μM of H2DCFH-DA for 25 min at 37°C. The increase in fluorescence due to production of ROS was noted (excitation 488 nm, emission 529 nm) using a fluorimeter. In another set of experiment, cells were incubated with NAC (0, 2.5, 5, 10 and 25 mM), 1 h prior to treatment with complex 3 (10 μM) for 24 h and the fluorescence increase because of production of ROS was noted (excitation 488 nm, emission 529 nm) using a fluorimeter. Further, MTT assay was done on cells incubated with NAC (0 and 10 mM) prior to addition of complex 3 (0 and 10 μM) for 24 h.

To measure mitochondrial membrane potential (MMP), B16F10 cells treated with complex 3 (0 and 10 μM) for 24 h were incubated with JC-1(5,5′,6,6′-tetrachloro-1, 1′, 3, 3′-tetraethylbenzimidazolylcarbocyanine iodide, Sigma). The reagent gives fluorescence in the FITC channel for green monomers in case of healthy cells having high mitochondrial membrane potential and in the PE-Texas Red A channel for red aggregates signifying apoptotic cells indicating a drop in the mitochondrial membrane potential. Data from 10⁶ cells were analyzed in a BD LSRFortessa cell analyzer for each sample.

Moreover, B16F10 cells treated with complex 3 (0, 2.5, 5, 10 and 25 μM) for 24 h were incubated with Rhodamine 123 and the fluorescence emission was measured in a spectrofluorometer (LS50B; PerkinElmer) using fluorescence intensity at 535 nm as described earlier [29].

B16F10 cells were treated with complex 3 (0, 2.5, 5, 10 and 25 μM) for 24 h, and the level of cytosolic cytochrome c was determined by a cytochrome c colorimetric assay kit, as described earlier [13].

B16F10 cells were treated with complex 3 (0 and 10 μM) for 24 h and confocal microscopy for immunocytochemistry analysis of p53 and p21 was done as reported earlier [13]. Cells were observed under an Andor spinning disk confocal microscope and images were acquired.

Western blotting of the lysates of the cells treated with complex 3 (0, 2.5, 5 and 10 μM) for 24 h was performed using 10–15% SDS-PAGE gels, primary antibodies, alkaline phosphatase conjugated secondary antibodies and NBT-BCIP as a chromogenic substrate as described earlier [30]. The experiments were done for the detection of PARP cleavage and for determining expression levels of pro-caspase 3 and pro-caspase 9 along with cytosolic cytochrome c and Bax. The expressions of p53, p-p53 (ser 15), p21 and Bax were determined in cells treated with complex 3 (0, 5 and 10 μM) for 24 h. In another set of experiments, cells were pretreated with 15 and 30 μM of the p53 transactivation inhibitor pifthrin-α (PFT–α), then treated with complex 3 (10 μM) for 24 h and analyzed for the level of expressions of p53, p21, Bax and cytochrome c. MTT assay was conducted to determine the growth inhibitory action of complex 3 (0 and 10 μM) in presence of PFT-α (0, 5, 15 and 30 μM). Also cells treated with NAC (0, 5 and 10 mM) for 1 h and then treated with complex 3 (10 μM) for 24 h were analyzed for the level of p53 and p-p53 (ser 15) expression.

In order to select the least toxic doses, male BALB/c mice were administered (i.p.) with 0, 5, 10, 20 and 40 mg/kg body weight of complex 3, observed for 60 days, and inoculated with B16F10 cells as described earlier [31]. Briefly, mice inoculated with 5 × 10⁴ B16F10 cells in the right hind limb (subcutaneously) were monitored for 15 days, after which complex 3 (5 and 10 mg/kg mouse body weight) was administered by intraperitoneal (i.p.) injection. The final dose was given on the 23^rd day and the mice were sacrificed on the 24^th day. Care and maintenance of animals were done in adherence to the guidelines of the Institutional Animal Care and Use Committee.

Slides fixed in 4% paraformaldehyde were incubated with primary antibodies for p53, p21, NF-κB p65 and p50, VEGF and MMP-9 overnight at 4°C. After washing with 1X TBST, slides were incubated with secondary antibodies for the above proteins for 2 h and stained with DAPI for identification of the nucleus. Cells were observed under an Andor spinning disk confocal microscope and images were acquired.