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Synergistic inhibition of melanoma xenografts by Brequinar sodium and Doxorubicin
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Synergistic inhibition of melanoma xenografts by Brequinar sodium and Doxorubicin Mathura Subangari Dorasamya,b, Aravind ABc, Kavitha Nellorec, Pooi-Fong Wonga,
T
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a
Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia Aurigene Discovery Technologies, IPPP, University Malaya, Kuala Lumpur, 50603, Malaysia c Aurigene Discovery Technologies Limited, Electronic City, Bangalore, 560100, Karnataka, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: A375 Brequinar Doxorubicin In vivo Cyclin B1 pcdc-2 p21
Malignant melanoma continues to be a fatal disease for which novel and long-term curative breakthroughs are desired. One such innovative idea would be to assess combination therapeutic treatments – by way of combining two potentially effective and very different therapy. Previously, we have shown that DHODH inhibitors, A771726 and Brequinar sodium (BQR) induced cell growth impairment in melanoma cells. Similar results were seen with DHODH RNA interference (shRNA). In the present study, we showed that combination of BQR with doxorubicin resulted in synergistic and additive cell growth inhibition in these cells. In addition, in vivo studies with this combination of drugs demonstrated an almost 90% tumor regression in nude mice bearing melanoma tumors. Cell cycle regulatory proteins, cyclin B1 and its binding partner pcdc-2 and p21 were significantly downregulated and upregulated respectively following the combined treatment. Given that we have observed synergistic effects with BQR and doxorubicin, both in vitro and in vivo, these drugs potentially represent a new combination in the targeted therapy of melanoma.
1. Introduction Malignant melanoma is a melanocyte neoplasm that usually takes place in the skin and occasionally harbors mutations in genes including BRAF [1,2]. Each year, the number of estimated new cases of malignant melanoma is 132,000 and approximately 48,000 patients die from malignant melanoma globally. When malignant melanoma is diagnosed at the early stage (stage 0/I), the patient’s 5-year survival rate is more than 90% after surgical excision. However, when early detection fails, it tends to invade deeply and metastasize to lymph nodes and other organs. Median overall survival rate for melanoma patients with metastasis is less than 1 year [3]. The treatment of metastatic melanoma is rapidly evolving. The potent and specific BRAF inhibitors vemurafenib and dabrafenib, as compared with cytotoxic chemotherapeutic drugs, have significantly improved response rates, along with progression-free and overall survival in patients with metastatic melanoma with BRAF V600E or V600K mutations [4,5]. However, acquired resistance to BRAF inhibitors frequently develops through reactivation of the mitogen-activated protein kinase (MAPK) pathway, resulting in a median progression-free survival of 6 to 8 months [5–8]. In addition, the use of BRAF inhibitors may result in the development of secondary skin tumors, originating from a paradoxical activation of the MAPK pathway
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in cells without a BRAF mutation [4,9–14]. Given that the majority of BRAF inhibitor resistance occurs through reactivation of MAPK, several potent, non-ATP competitive MEK inhibitors were developed (eg, trametinib and cobimetinib) [15] that show no off-target effects on other kinases and are currently being used in clinical trials. MEK inhibitors are able to target MAPK-dependent tumors and exhibit distinct efficacies against BRAF- and KRAS-mutant melanomas [16,17]. Studies reveal that BRAF inhibitor-resistant tumor cells are highly sensitive to MEK inhibition and demonstrate that targeted pharmacological MEK inhibition may be a highly effective therapeutic alternative in BRAF inhibitor-resistant melanoma [17]. In light of these findings, focus has turned to dual inhibition of BRAF and its downstream target, MEK [18]. The results of combination therapy of MEK inhibitors and BRAF inhibitors are promising. New clinical trials confirm improved PFS progression-free survival with concurrent inhibition of BRAF and MEK. A Phase III, randomized study, compared patients with BRAF-mutated melanomas treated with vemurafenib 960 mg twice daily versus vemurafenib plus cobimetinib 60 mg daily for 21 days followed by 7 days off. The group on combination therapy displayed prolonged median PFS (9.9 months vs 6.2 months). The latest Phase III clinical trial of dabrafenib (BRAF inhibitor) and trametinib versus monotherapy with vemurafenib showed increased median PFS (11.4 months vs 7.3 months;
Corresponding author. E-mail address: [email protected] (P.-F. Wong).
https://doi.org/10.1016/j.biopha.2018.11.010 Received 1 September 2018; Received in revised form 2 November 2018; Accepted 2 November 2018 0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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antagonistic by taking into account the entire shape of the growth inhibition curve [23]. The resulting combination index (CI) theorem of Chou-Talalay offers quantitative definition for additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations.
HR 0.56; 95% confidence interval [CI]: 0.46– 0.69; P = 0.001) and improved overall survival at 12 months with combination therapy (72% vs 65%; HR 0.69; 95% CI: 0.53–0.89; P = 0.005) [19]. Despite these clinical benefits, the emergence of resistance to BRAF inhibitors and eventually to MEK inhibitors restricts the therapeutic efficacy of these kinase inhibitors. In an attempt to overcome this resistance, more potential molecules targeting other aberrant signalling pathways are in clinical development [20,21]. We have previously shown that DHODH inhibitors such as A771726 (Leflunomide metabolite) and Brequinar sodium (BQR) expose their anti-proliferative effects on melanoma, myeloma and lymphoma cells. These inhibitors arrested cancer cells growth in S-phase, decreased cMyc and increased p21 protein expression [22]. Furthermore, DHODH suppression by shRNA affected cell proliferation, c-Myc and its target protein, p21 in melanoma cells. In this study, we sought to investigate the effect of combining BQR and doxorubicin in vitro and in human melanoma xenograft. The combination treatment further sensitized the in vitro and in vivo melanoma tumor growth.
2.5. In vivo toxicity assessment of BQR and doxorubicin MTD is defined as the highest tolerable dose which does not produce major life threatening toxicity for the study duration [24]. Toxicity assessment of BQR and doxorubicin were performed using CD-1 mice, which are commonly used in toxicology and cancer research [25–27]. The MTD doses in this study were selected based on previous findings of the efficacy of BQR and doxorubicin in CD-1 mice [43–46]. Single dose of BQR (20 mg/kg) and doxorubicin (1 mg/kg) as well as in combination: BQR + doxorubicin (10 mg/kg + 1 mg/kg), BQR + doxorubicin (20 mg/kg + 1 mg/kg) were tested. Toxicity study was conducted in 5 mice per group for 14 days. Drugs were administered to CD-1 mice orally or intravenously (lateral tail vein) and toxicity was evaluated based on clinical symptoms such as lethargy, loss of body weight, diarrhoea and behavioural changes. All animal experiments were performed in accordance with the animal care and use protocol (Aurigene/ IAEC/PCD/018 E-13/09-2014) approved by the Institutional Animal Care and Use Committee, Aurigene Discovery Technologies.
2. Materials and methods 2.1. Cells Human melanoma (A375) cell line was obtained from American Type Cell Collections. A375 cells were grown in DMEM (Sigma, USA). The medium was supplemented with 10% heat-inactivated fetal bovine serum (Sigma, USA), 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco, USA). Cells were maintained in a 5% CO2 atmosphere at 37 °C.
2.6. Establishment of tumor xenograft Athymic nude mice, aged 5 to 6 weeks, and weighing approximately 18–20 g from Harlan laboratories (Hyderabad, India) were housed in individually ventilated cages and were fed ad libitum with reverse osmosis treated water and gamma irradiated rodent pellet. A375 xenografts were established by subcutaneous injection of 5 × 106 A375 cells suspended in 150 μl of 1:1 ratio of Hank’s balanced salt solution and extracellular matrix. The mixture was injected onto the right flank of the mice. The individual animal body weights were recorded daily before the administration of test items and the tumor volumes were measured thrice weekly. All animal experiments were performed in accordance with the animal care and use protocol (Aurigene/IAEC/ PCD/018 E-13/09-2014) approved by the Institutional Animal Care and Use Committee, Aurigene Discovery Technologies.
2.2. Drugs and antibodies A771726, BQR and PLX 4720 were pharmaceutical standard with a purity of 99.50%, 98.80% and 99.55%, respectively and these drugs were synthesized at Aurigene Discovery Technologies Limited, Bangalore, India. Doxorubicin was purchased from Celon Labs, India. Antibodies for cyclin B1, pcdc-2 and p21 and loading control, GAPDH were procured from Cell Signaling Technology, USA. Secondary antibody, anti-rabbit polyclonal antibody was secured from Sigma, USA. 2.3. Cell proliferation assay
2.7. Assessment of the effect of combined drug treatments in tumor xenograft
A375 cells were seeded in 96-well plates at 1000 cells per well. The DHODH inhibitors - A771726 and BQR were dissolved in Hybrimax DMSO (Sigma, USA) whereas doxorubicin was dissolved in sterile distilled water. The additive or synergy effect between BQR and doxorubicin was determined using a non-fixed ratio method in which fixed concentrations of doxorubicin (0.3, 0.15, 0.075, 0.00375, 0.001875 and 0.009375 μM) were added with increasing concentrations of BQR (30.0, 10.0, 3.33, 1.11, 0.37, 0.123, 0.041 and 0.014 μM) and incubated for 72 h. Each plate contained control wells for vehicle (0.5% DMSO), BQR alone and doxorubicin alone in triplicates. Cell viability was determined using Alamar blue assay. Cells were treated with 50 μl/well of 1 mg/ml resazurin (Sigma, USA) dissolved in PBS (Sigma, USA). The fluorescence reading was measured at Ex/Em of 531/595 nm. Cell viability was calculated as a percentage of fluorescence or absorbance measured in the treated wells relative to the DMSO control wells. Experiments were repeated three times and mean ± SE was calculated.
When the mean tumor volume reached 100–150 mm3 in size, animals were randomized based on tumor volumes into seven groups and allocated nine animals for each group. The treatment groups are as following: (i) Vehicle control (diluent control), (ii) BQR (10 mg/kg), (iii) BQR (20 mg/kg), (iv) Doxorubicin (1 mg/kg), (v) BQR + Doxorubicin (10 mg/kg + 1 mg/kg), (vi) BQR + Doxorubicin (20 mg/kg + 1 mg/kg) and (vii) PLX 4720 (Vemurafenib), a BRAF inhibitor (20 mg/kg). BQR was formulated in 0.5% CMC + 0.25% tween 20 and administered orally once every alternate day, Doxorubicin was formulated in 5% dextrose and administered intravenously once every alternate day. Whereas, PLX 4720 was formulated in 5% DMSO + 1% methyl cellulose and administered orally once per day. The formulations were prepared fresh and used immediately. Body weights were monitored every day and tumor volumes were measured thrice weekly. The tumor volumes were measured using the formula: [A (length) x B (width)2]/2 [28]. The experiment was stopped at day 14, and all mice were sacrificed. The percentage of tumor growth inhibition was calculated using the following formula:
2.4. Assessment of the effect of combined drug treatments in vitro The effect of combined drug treatment of BQR and doxorubicin were examined using the median effect analysis method described by Chou and Talalay [23]. Calculation of combination index (CI) was performed using Compusyn software (Chou-Talalay, ComboSyn) to determine whether a combination is synergistic, additive, or
[vehicle control TV (day n – day 1)] – [drug TV (day n – day 1)]/[vehicle control TV (day n – day 1)] x 100, where TV = tumor volume and n = day of treatment 30
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evaluated for their anti-tumor activity in A375 xenograft model in athymic nude mice. PLX 4720 (Vemurafenib), a BRAF inhibitor approved for the treatment of late-stage melanoma [29] was used as the positive control. After 14 days of treatment, the tumor volume of mice administered with vehicle was 1090.0 ± 134.7 mm3 (Fig. 3A). By contrast, the tumor volumes of mice -administered with 20 mg/kg BQR; 10 mg/kg BQR + 1 mg/kg doxorubicin; 20 mg/kg BQR + 1 mg/kg doxorubicin and PLX 4720 were significantly lower than those of the vehicle control animals with tumor volumes of 404.6 ± 105 mm3; 388.6 ± 55.6 mm3; 217.2 ± 52.1 mm3 and 287.8 ± 50.7 mm3, respectively (Fig. 3A). However, there were no significant differences in the tumor volume of animals administered with single agent in comparison to the combination drugs as well as for those animals given combination drugs in comparison with PLX 4720. This is likely because of the large variances in the tumor volume between groups. To examine further the effectiveness of the drug treatment, the percentage tumor growth inhibition (TGI) was calculated relative to the vehicle control group. At the end of 14 days treatment, mice treated with 10 mg/kg and 20 mg/kg BQR as a single agent reduced tumor growth by 52.7 ± 6.5% and 70.6 ± 10.5% respectively, whereas doxorubicin at 1 mg/kg single agent suppressed tumor growth by only 36.1 ± 4.7% relative to the vehicle control (Fig. 3B). The positive control drug, PLX 4270 resulted in tumor growth inhibition (TGI) of 82.7 ± 4.6% relative to the vehicle control. The percentage TGI with 10 mg/kg BQR + 1 mg/kg doxorubicin (72.2 ± 5.2%) was significantly higher than that of treatment with 10 mg/kg BQR alone (52.7 ± 6.5%). Similarly, the percentage TGI with 20 mg/kg BQR + 1 mg/kg doxorubicin (89.9 ± 4.5%) was significantly higher than that of the treatment with 20 mg/kg BQR alone (70.6 ± 10.5%). The combination of 10 mg/kg BQR with 1 mg/kg doxorubicin, however, yielded a TGI of 72.2 ± 5.2% only compared to the TGI of PLX 4270 (82.7 ± 4.6%). Nevertheless, the combination of 20 mg/kg BQR with 1 mg/kg doxorubicin (89.9 ± 4.5%) resulted in higher percentage TGI compared to PLX 4270 (82.7 ± 4.6%). Analysis with Compusyn showed that doxorubicin co-administered with BQR at higher dosage synergistically inhibited tumor growth (Fig. 3C). This combination not only inhibited tumor growth more effectively but also reduced vascularity in the tumor compared to vehicle control, BQR, doxorubicin and PLX 4720 alone, (Fig. 4A and B). The maximal body weight change of the treated mice was below 10% compared to the vehicle control treated mice, indicating a lack of cumulative toxicity during treatment (Fig. 5).
2.8. Tumor lysate protein extraction and immunoblotting Excised tumors were minced and suspended in tissue protein extraction reagent (Cell lysis buffer) (Cell Signaling Technology, USA) supplemented with protease and phosphatase inhibitor cocktails (Sigma, USA) and homogenized. The homogenized tissue was transferred to a pre-chilled microcentrifuge tube, incubated on ice for 10 min and then centrifuged at 15,000 x g for 20 min at 4 °C. The supernatant containing total cellular proteins was collected for immunoblotting analysis. Extracted protein was quantified with Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Equal amount of protein was subjected to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (GE Healthcare, USA). The membrane was blocked with Tris-buffer saline with 0.1% Tween-20 (TBST) containing 5% dry milk (Cell Signaling, USA). After blocking, the membrane was probed with appropriate primary antibodies overnight at 4 °C. On the next day, the binding of the primary antibodies was detected with anti-rabbit polyclonal secondary antibody labelled with horseradish peroxidase. The blot was developed with ECL Western blotting detection system (Pierce, USA) and subsequently the membrane was exposed to X-ray film. The intensity of each protein band was measured with ImageJ. 2.9. Statistical analysis Statistical significance of the differences between control and treatment was analyzed with Student’s t-test and one-way ANOVA with Dunnett’s or Tukey’s Multiple Comparison Tests, where applicable using GraphPad Prism 6.0. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 were considered statistically significant. 3. Results 3.1. BQR in combination with doxorubicin enhanced melanoma inhibition A375 cells were incubated with BQR or A771726 in combination with doxorubicin for 72 h. Synergistic or additive effects were achieved in most combinations except for BQR with 0.009375 μM doxorubicin. Combination of BQR with doxorubicin (Fig. 1A and B) showed greater dose-dependent proliferation inhibition than BQR alone (approximately 56.25 ± 2.6% inhibition at 30 μM) and doxorubicin alone (approximately 83.25 ± 4.33% at 0.3 μM) in A375 cells. Significant proliferation inhibition was achieved with 30 μM BQR in combination with 0.075 μM; 0.15 μM and 0.3 μM doxorubicin, with the percentage of inhibition of approximately 78.0 ± 6.03%; 91.25 ± 4.87% and 97.25 ± 0.63%, respectively in comparison to treatment with BQR alone. Whereas, the percentages of inhibition for treatment with 0.3 μM doxorubicin in combination with 30 μM and 10 μM BQR were approximately 97.25 ± 0.63 and 96.75 ± 0.85%, respectively.
3.4. Combination of brequinar and doxorubicin treatment target cyclin B1, pcdc-2 and p21 in A375 xenografts Previously, we have shown that treatment with BQR primarily induced S-phase and G2/M cell cycle arrest and p21 protein upregulation in vitro. Here, we showed that single treatments of BQR downregulated cyclin B1 expression by approximately 1.9-fold when compared to the vehicle control group. This protein was further decreased with the combination of 10 or 20 mg/kg BQR + 1 mg/kg doxorubicin as illustrated in Fig. 6A. Notably, the combination treatments significantly suppressed the expression of cyclin B1 by 2-2.4-fold compared to the single treatment of 20 mg/kg BQR; by 10.9–12.2-fold compared to doxorubicin treatment alone and by 3.4-3.8-fold compared to vehicle control (Fig. 6A). Interestingly, treatment with doxorubicin and PLX 4720 alone did not reduce the expression of this protein. To understand further, the binding partner of cyclin B1, pcdc-2 expression was also examined. The data presented in Fig. 6B shows pcdc-2 expression was downregulated in all treatment groups except doxorubicin when compared to the vehicle control group. The expression of pcdc2 was markedly downregulated by 5-20-fold in the presence of 10 mg/kg BQR + 1 mg/kg doxorubicin as compared to its respective single agents and vehicle control. Similarly, this downregulated trend of pcdc2 expression was observed in the combination treatment of 20 mg/
3.2. BQR in combination with doxorubicin was well tolerated in mice In vivo toxicity assessment of the single and combination of BQR and doxorubicin drugs was performed in CD-1 mice for a period of 14 days and mice weight was recorded (Fig. 2A). The tested doses of BQR and doxorubicin were well tolerated and no treatment related clinical signs or mortality was observed. Non-treated mice demonstrated a weight gain of 8% at the end of day 14. Instead of weight loss, mice with single treatment displayed a weight gain between 4–4.6% whereas mice with combination treatment saw a weight gain of no more than 1.4% (Fig. 2B). 3.3. Brequinar sodium potentiates anticancer effect of doxorubicin in A375 xenografts The in vitro synergistic effect of BQR and doxorubicin was further 31
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Fig. 1. BQR in combination with doxorubicin synergistically exerts inhibition of A375 melanoma cells. A375 cells were treated with BQR or doxorubicin alone or in combination for 72 h. Inhibition of proliferation was measured by Alamar blue assay. A, Percentage of proliferation inhibition by BQR (30.0, 10.0, 3.33, 1.11, 0.37, 0.123, 0.041 and 0.014 μM) in combination with doxorubicin (0.3, 0.15, 0.075, 0.00375, 0.001875 and 0.009375 μM). B, Percentage of proliferation inhibition of doxorubicin (0.3, 0.15, 0.075, 0.00375, 0.001875 and 0.009375 μM) in combination with BQR (30.0, 10.0, 3.3, 1.1 and 0.37 μM). Values shown are mean ± SE of 4 independent experiments. **P < 0.01 and ****P < 0.0001 (One-way ANOVA, Dunnett’s Multiple Comparison Tests). Brequinar and doxorubicin are abbreviated as BQR and Doxo, respectively.
4. Discussion
kg BQR + 1 mg/kg doxorubicin, albeit insignificant. The expression of the cell cycle regulatory protein, p21 in all single except doxorubicin and combination treatments were decreased as compared to the vehicle control. Nevertheless, as hypothesized, the expression of this protein was significantly upregulated by 0.3–0.5-fold in both the combination treatments as compared to treatment with BQR alone (Fig. 6C). Intriguingly, this protein was not detected with PLX 4720 (Fig. 6C and D).
Acknowledged as one of the most aggressive skin cancers, melanoma has limited therapeutic options and its occurrence is steadily and rapidly increasing. While this is true, there has been a host of discoveries and applications of new targeted therapy agents that have brought about significant benefits. Nevertheless, resistance to chemotherapy as well as undesirable side effects remain as a monumental challenge in the clinical treatment of malignant melanoma [30]. Past studies using cell lines derived from metastatic lesions resulted in cells becoming resistant to both MEK and PI3K inhibitors when grown in 3D Fig. 2. Effect of drug treatment on CD1 mice. A, Body weight of CD-1 mice during drug treatment. B, The percentage of body weight gain after 14 days of treatment in CD-1 mice. Mice were weighed daily and the percentage of body weight gain was obtained by subtracting mice weight three days before treatment from the weight on day 14. Brequinar and doxorubicin are abbreviated as BQR and Doxo, respectively.
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Fig. 3. Combination of BQR and doxorubicin achieved greater tumor inhibition in A375 melanoma xenograft. A375 xenograft mice were treated with vehicle control, BQR (10 mg/kg or 20 mg/kg, oral, every alternate day), doxorubicin (1 mg/kg, i.v. every alternate day) and PLX 4720 (20 mg/kg, oral, daily). Tumor volumes were measured once every 3 days. A, Data show mean tumor volume (mm3) ± standard error mean (SEM, error bars) of 9 mice per group. B, Percentage of tumor growth inhibition (TGI) ± standard error and CI values. TGI were analysed with the Compusyn software to show additive/synergistic effects. CI = 0.80 indicates additive effects, whereas CI = 0.44 indicates synergism. *P < 0.05 and **P < 0.01 relative to vehicle control (One-way ANOVA, Dunnett’s Multiple Comparison Test). # P < 0.05 and ###P < 0.001 relative to the respective single agent (One-way ANOVA, Tukey’s Multiple Comparison Test). Brequinar and doxorubicin are abbreviated as BQR and Doxo, respectively.
dabrafenib lasts only 6–8 months before the disease relapses especially in patients with metastatic melanoma [38]. With that in mind, rational combination approaches are strongly preferred in order to improve the overall patient progression-free survival (PFS), overcome or delay the development of multi-drug resistance and reduce the incidents of side effects [39–42]. In the present study, the anti-tumor effect of BQR alone and in combination with doxorubicin, an inhibitor of topo-isomerase 2 [43] was investigated. Preclinical studies of the maximum tolerated dose of BQR in several animal species have been reported previously. For instance, a dose of 193 mg/m2 per day induced a 10% lethality in mice for a period of 5 days every 4 weeks. Furthermore, preclinical studies in dogs showed that they were only able to tolerate a maximum dose of 6 mg/m2 per day on the same schedule [44]. Today, phase I of clinical trials with anticancer drugs recommend dosages for humans to be one third of that (13) or 2 mg/m2. However, previous reports have stated that doses of 100 mg/m2 (i.v.) daily for 5 days every 4 weeks had been safely administered to patients with advanced cancer and minimal prior treatment [45]. Building on from this knowledge, 2 doses of BQR were selected for this study, 10 mg/kg and 20 mg/kg. Doxorubicin is a widely used antineoplastic agent for many cancer types. However, it is ineffective against melanoma cells due to the frequent development of resistance [46–48]. In addition to its role as a RNA-synthesis inhibitor, He et al. [49] reported that doxorubicin significantly reduced both protein and mRNA expression of DHODH in U1690 cells in a concentration-dependent manner. Further to this,
culture. Similar outcome was also observed in in vivo studies, where MEK inhibitor, AZD6244 led to the stabilisation of established human melanoma xenografts, but not tumor regression [31]. In other words, as the melanomas progress, there seem to be functional redundancy between the various signalling pathways in these cells [32]. Hence, targeting multiple signalling pathways simultaneously could be a better strategy to induce melanoma regression. We have previously shown that Brequinar (BQR) produces antiproliferative effect on melanoma cells by inducing cell cycle arrest via the DHODH-independent pathway that is associated with p21 up-regulation and c-Myc down-regulation in vitro. In addition, we have also demonstrated that exogenous uridine was able to completely reverse the anti-proliferative effect mediated by BQR at all concentrations [22]. These results strongly suggest that two mechanisms, DHODH-independent and DHODH-dependent may possibly be involved in the anti-proliferative effect of BQR on A375 cells. Therefore, targeting multiple inhibitory pathways may improve clinical efficacy. The FDA approved melanoma inhibitors such as pembrolizumab targeting the PD-1 (programmed cell death protein 1, ipilumab targeting the CTLA-4, cytotoxic T lymphocyte-associated antigen 4, MEK inhibitor, trametinib and BRAF inhibitors, vemurafenib and dabrafenib represent an important milestone in more effective treatment of advanced melanoma [4,19,33–36]. Nevertheless, it is clear that the clinical use of these single-agent therapies have limitations. For example, ipilimumab only showed 4.5% objective response rate when used alone in a Phase II clinical trial [37]. The efficacy of vemurafenib and 33
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Fig. 4. Combination of BQR and doxorubicin achieved greater therapeutic effect in melanoma xenograft in vivo. A, Representive images of the location of tumor xenografts in atymic nude mice and B, tumor regression at the end of the study. Brequinar and doxorubicin are abbreviated as BQR and Doxo, respectively.
with multiple drugs (combined treatment) has been actively discussed and embraced in recent years. Brown et al. demonstrated that a combination of leflunomide and doxorubicin sensitized xenograft tumors derived from MDA-MB-231 cell lines. Moreover, our in vivo studies used a dosage of doxorubicin (1 mg/kg) that is approximately 0.1 times the recommended human dose based on body surface area [52]. The dosing schedule in this study is fairly short in comparison to various other reports. It is one that has been designed to achieve a prolonged drug exposure in an outpatient clinical setting, potentially yielding superior antitumor results. To further elucidate the underlying mechanism of melanoma xenograft inhibition, we analysed the expression profiles of important cell cycle regulator proteins such as p21, cyclin B1 and p-cdc-2. Interestingly, a clear and significant downregulation was seen in the expression of cyclin B1 and p-cdc-2 in the combined treatment. Correspondingly, the cell-cycle regulatory protein, p21 was observed to be substantially upregulated in both the combined treatments. In the cell cycle of eukaryotes, activation of cyclin B1-cdk 1 (cyclin dependent kinase 1) also known as cdc-2 brings the onset of mitosis. Cyclin B1 is involved in the transition from G2 to M phase however it becomes unregulated in cancer cells where it is often found overexpressed. Overexpression of cyclin B1 thus leads to uncontrolled cell growth [53–59]. Alternatively, cdc-2 phosphorylation inhibits cell differentiation at the G2/M phase that leads to an arrest in the entry of eukaryotic cell into mitosis [60]. The suppression of cell proliferation by upregulation of p21 expression is a prominent mechanism activated by several chemotherapy drugs that cause genotoxic stress [61]. As determined in this study, the suppressed expression of cyclin B1 and pcdc-2 with concomitant increase in the expression of p21 protein especially in the combined treatment further reaffirms our earlier in vitro findings [22] which suggest that these inhibitors arrest cancer cell proliferation primarily at S-phase and G2/M phase. Nevertheless, our observation of a reduction in p21 expression in all treatments compared to vehicle control in general, but an increase with combination treatment, leads us to the possibility of the dual role of p21 in oncogenesis as described by Cmielova et al. [62]. They suggest that the expression of p21 in cancer
Fig. 5. Percentage of body weight change in A375 xenograft mouse model during the treatment period. Mice were weighed daily and their body weight was expressed as a percentage of body weight change. Brequinar and doxorubicin are abbreviated as BQR and Doxo, respectively.
various studies have also reported that doxorubicin in combination with inhibitors such as berberine (in mouse xenograft) [50] and benzodiazepinedione [51] demonstrated a better therapeutic effect than either single drug alone. Our findings show that BQR in combination with doxorubicin synergistically suppressed the growth of A375 cells both in vitro and in vivo within 14 days of prolonged exposure to these inhibitors without any noticeable toxic profiles. These results provide the first in vitro and in vivo evidence that BQR co-administered with doxorubicin can potentiate the RNA-synthesis inhibitor effect via the DHODH pyrimidine pathway and as such, may provide promising therapeutic benefit as a combination therapy for melanoma. The rationale of targeted therapy 34
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Fig. 6. Combined treatment with 20 mg/kg BQR and 1 mg/kg doxorubicin resulted in greater downregulation of cyclin B1 and pcdc-2 in A375 melanoma xenograft. A, B and C, shows quantification of cyclin B1, pcdc-2 and p21, respectively whereas D, displays representative immunoblots of 4 animals of all 7 groups of mice. All the treated and non-treated groups of A375 melanoma xenografts were sacrificed on day 14 and tumors were excised. Tumor powder was prepared with liquid nitrogen and the protein was digested with cell lysis buffer. Protein quantification was performed with BCA kit and Western blot analysis was carried out. Equal amount of protein lysates were used with the antibodies indicated. GAPDH served as protein loading control. Data represents standard error mean ± (SEM, error bars) of 9 mice per group *, P < 0.05, **P < 0.01 and ***P < 0.001.
Declarations of interest
cells depends on the extent of the DNA damage. Low-level DNA damage leads to high expression of p21 and it induces cell cycle arrest whereas extensive DNA damage decreases the amount of p21 and the cancer cells undergoes apoptosis. As such, the role of p21 is context dependent as it can act as pro-apoptotic or anti-apoptotic in different conditions [63]. With this in mind, these results suggest that the upregulation of p21 is a pivotal mechanism for the enhanced suppression of melanoma cells.
None. Acknowledgement I would like to thank all Aurigene Discovery Technologies staff for their guidance and support. References
5. Conclusion
[1] E.D. Deeks, Nivolumab: a review of its use in patients with malignant melanoma, Drugs 74 (11) (2014) 1233–1239. [2] R. Okura, et al., Expression of AID in malignant melanoma with BRAFV600E mutation, Exp. Dermatol. 23 (5) (2014) 347–348. [3] E. Mashima, et al., Nivolumab in the treatment of malignant melanoma: review of the literature, Oncol. Ther. 8 (2015) 2045. [4] P.B. Chapman, et al., Improved survival with vemurafenib in melanoma with BRAF V600E mutation, N. Engl. J. Med. 364 (26) (2011) 2507–2516. [5] A. Hauschild, et al., Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial, Lancet 380 (9839) (2012) 358–365. [6] D.B. Solit, N. Rosen, Resistance to BRAF inhibition in melanomas, N. Engl. J. Med. 364 (8) (2011) 772–774. [7] H. Shi, et al., Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy, Cancer Discov. 4 (1) (2014) 80–93. [8] E.M. Van Allen, et al., The genetic landscape of clinical resistance to RAF inhibition in melanoma, ASCO Annual Meeting Proceedings, (2013). [9] K.T. Flaherty, et al., Inhibition of mutated, activated BRAF in metastatic melanoma,
Findings from the present study revealed combination treatment of BQR and doxorubicin resulted in significant inhibitory effect on the growth of melanoma cells both in vitro and in vivo. The favourable synergistic effect and low toxic profiles of this treatment combination in melanoma xenograft model suggests that this combination has high potentials as targeted therapy for melanoma and warrants future pharmacokinetics and resistance studies. Funding This work was supported by Aurigene Discovery Technologies, India and University of Malaya Postgraduate Research Fund (PPP) Grant Number: PG007-2014B. 35
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[36] C. Garbe, S. Abusaif, T.K. Eigentler, Vemurafenib, Recent Results Cancer Res. 201 (2014) 215–225. [37] E.M. Hersh, et al., A phase II multicenter study of ipilimumab with or without dacarbazine in chemotherapy-naive patients with advanced melanoma, Invest. New Drugs 29 (3) (2011) 489–498. [38] J.A. Sosman, et al., Survival in BRAF V600–mutant advanced melanoma treated with vemurafenib, N. Engl. J. Med. 366 (8) (2012) 707–714. [39] B. Al-Lazikani, P. Workman, Unpicking the combination lock for mutant BRAF and RAS melanomas, Cancer Discov. 3 (1) (2013) 14–19. [40] S. Hu-Lieskovan, et al., Combining targeted therapy with immunotherapy in BRAFmutant melanoma: promise and challenges, J. Clin. Oncol. 32 (21) (2014) 2248–2254. [41] C. Nijenhuis, et al., Is combination therapy the next step to overcome resistance and reduce toxicities in melanoma? Cancer Treat. Rev. 39 (4) (2013) 305–312. [42] M. Voskoboynik, H.-T. Arkenau, Combination therapies for the treatment of advanced melanoma: a review of current evidence, Biochem. Res. Int. 2014 (2014). [43] D.A. Gewirtz, A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin, Biochem. Pharmacol. 57 (7) (1999) 727–741. [44] D.A. Noe, et al., Phase I and pharmacokinetic study of Brequinar sodium (NSC 368390), Cancer Res. 50 (15) (1990) 4595–4599. [45] C.L. Arteaga, et al., Phase I clinical and pharmacokinetic trial of Brequinar sodium (DuP 785; NSC 368390), Cancer Res. 49 (16) (1989) 4648–4653. [46] M. Panneerselvam, R. Bredehorst, C.W. Vogel, Resistance of human melanoma cells against the cytotoxic and complement-enhancing activities of doxorubicin, Cancer Res. 47 (17) (1987) 4601–4607. [47] M.G. Smylie, et al., A phase II, open label, monotherapy study of liposomal doxorubicin in patients with metastatic malignant melanoma, Invest. New Drugs 25 (2) (2007) 155–159. [48] D.A. Vorobiof, et al., Phase II study of pegylated liposomal doxorubicin in patients with metastatic malignant melanoma failing standard chemotherapy treatment, Melanoma Res. 13 (2) (2003) 201–203. [49] T. He, et al., Inhibition of the mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase by doxorubicin and brequinar sensitizes cancer cells to TRAIL-induced apoptosis, Oncogene 33 (27) (2014) 3538–3549. [50] A. Mittal, S. Tabasum, R.P. Singh, Berberine in combination with doxorubicin suppresses growth of murine melanoma B16F10 cells in culture and xenograft, Phytomedicine 21 (3) (2014) 340–347. [51] H.K. Koblish, et al., Benzodiazepinedione inhibitors of the Hdm2: p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo, Mol. Cancer Ther. 5 (1) (2006) 160–169. [52] K.K. Brown, et al., Adaptive reprogramming of de novo pyrimidine synthesis is a metabolic vulnerability in triple-negative breast cancer, Cancer Discov. 7 (4) (2017) 391–399. [53] S.K. Banerjee, et al., Expression of cdc2 and cyclin B1 in Helicobacter pylori-associated gastric MALT and MALT lymphoma: relationship to cell death, proliferation, and transformation, Am. J. Pathol. 156 (1) (2000) 217–225. [54] K.A. Hassan, et al., Cyclin B1 overexpression and resistance to radiotherapy in head and neck squamous cell carcinoma, Cancer Res. 62 (22) (2002) 6414–6417. [55] H. Kawamoto, H. Koizumi, T. Uchikoshi, Expression of the G2-M checkpoint regulators cyclin B1 and cdc2 in nonmalignant and malignant human breast lesions: immunocytochemical and quantitative image analyses, Am. J. Pathol. 150 (1) (1997) 15–23. [56] P. Rudolph, et al., Differential prognostic impact of the cyclins E and B in premenopausal and postmenopausal women with lymph node-negative breast cancer, Int. J. Cancer 105 (5) (2003) 674–680. [57] J.C. Soria, et al., Overexpression of cyclin B1 in early-stage non-small cell lung cancer and its clinical implication, Cancer Res. 60 (15) (2000) 4000–4004. [58] A. Wang, et al., Overexpression of cyclin B1 in human colorectal cancers, J. Cancer Res. Clin. Oncol. 123 (2) (1997) 124–127. [59] M. Zhao, et al., Expression profiling of cyclin B1 and D1 in cervical carcinoma, Exp. Oncol. 28 (1) (2006) 44–48. [60] D.O. Morgan, Principles of CDK regulation, Nature 374 (6518) (1995) 131–134. [61] T. Abbas, A. Dutta, p21 in cancer: intricate networks and multiple activities, Nat. Rev. Cancer 9 (6) (2009) 400–414. [62] J. Cmielova, M. Rezacova, p21Cip1/Waf1 protein and its function based on a subcellular localization [corrected], J. Cell. Biochem. 112 (12) (2011) 3502–3506. [63] S. Liu, W.R. Bishop, M. Liu, Differential effects of cell cycle regulatory protein p21(WAF1/Cip1) on apoptosis and sensitivity to cancer chemotherapy, Drug Resist. Updat. 6 (4) (2003) 183–195.
N. Engl. J. Med. 363 (9) (2010) 809–819. [10] C. Robert, J.-P. Arnault, C. Mateus, RAF inhibition and induction of cutaneous squamous cell carcinoma, Curr. Opin. Oncol. 23 (2) (2011) 177–182. [11] J. Carnahan, et al., Selective and potent Raf inhibitors paradoxically stimulate normal cell proliferation and tumor growth, Mol. Cancer Ther. 9 (8) (2010) 2399–2410. [12] P.A. Oberholzer, et al., RAS mutations are associated with the development of cutaneous squamous cell tumors in patients treated with RAF inhibitors, J. Clin. Oncol. 30 (3) (2012) 316–321. [13] L. Boussemart, et al., Prospective study of cutaneous side-effects associated with the BRAF inhibitor vemurafenib: a study of 42 patients, Ann. Oncol. 24 (6) (2013) 1691–1697. [14] F. Su, et al., RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors, N. Engl. J. Med. 366 (3) (2012) 207–215. [15] K.D. Rice, et al., Novel carboxamide-based allosteric MEK inhibitors: discovery and optimization efforts toward XL518 (GDC-0973), ACS Med. Chem. Lett. 3 (5) (2012) 416–421. [16] G. Hatzivassiliou, et al., Mechanism of MEK inhibition determines efficacy in mutant KRAS-versus BRAF-driven cancers, Nature 501 (7466) (2013) 232–236. [17] D.B. Solit, et al., BRAF mutation predicts sensitivity to MEK inhibition, Nature 439 (7074) (2006) 358–362. [18] K.A. Tran, et al., MEK inhibitors and their potential in the treatment of advanced melanoma: the advantages of combination therapy, Drug Des. Devel. Ther. 10 (2016) 43. [19] C. Robert, et al., Improved overall survival in melanoma with combined dabrafenib and trametinib, N. Engl. J. Med. 372 (1) (2015) 30–39. [20] M. Hao, et al., Advances in targeted therapy for unresectable melanoma: new drugs and combinations, Cancer Lett. 359 (1) (2015) 1–8. [21] A. Niezgoda, P. Niezgoda, R. Czajkowski, Novel approaches to treatment of advanced melanoma: a review on targeted therapy and immunotherapy, Biomed Res. Int. 2015 (2015). [22] M.S. Dorasamy, et al., Dihydroorotate dehydrogenase inhibitors target c-Myc and arrest melanoma, myeloma and lymphoma cells at S-phase, J. Cancer 8 (15) (2017) 3086–3098. [23] T.-C. Chou, P. Talalay, Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors, Adv. Enzyme Regul. 22 (1984) 27–55. [24] S. Robinson, et al., A European pharmaceutical company initiative challenging the regulatory requirement for acute toxicity studies in pharmaceutical drug development, Regul. Toxicol. Pharmacol. 50 (3) (2008) 345–352. [25] S. Cui, C. Chesson, R. Hope, Genetic variation within and between strains of outbred Swiss mice, Lab. Anim. 27 (2) (1993) 116–123. [26] R. Chia, et al., The origins and uses of mouse outbred stocks, Nat. Genet. 37 (11) (2005) 1181–1186. [27] M.C. Rice, S.J. O’Brien, Genetic variance of laboratory outbred Swiss mice, Nature 283 (5743) (1980) 157–161. [28] D.S. Martin, et al., Therapeutic utility of utilizing low doses of N-(phosphonacetyl)L-aspartic acid in combination with 5-fluorouracil: a murine study with clinical relevance, Cancer Res. 43 (5) (1983) 2317–2321. [29] J. Tsai, et al., Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity, Proc. Natl. Acad. Sci. U. S. A. 105 (8) (2008) 3041–3046. [30] S. Liu, et al., Genetic variants in the genes encoding rho GTPases and related regulators predict cutaneous melanoma-specific survival, Int. J. Cancer 141 (4) (2017) 721–730. [31] N.K. Haass, et al., The mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor AZD6244 (ARRY-142886) induces growth arrest in melanoma cells and tumor regression when combined with docetaxel, Clin. Cancer Res. 14 (1) (2008) 230–239. [32] K.S. Smalley, et al., Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases, Mol. Cancer Ther. 5 (5) (2006) 1136–1144. [33] A. Ribas, et al., Pembrolizumab versus investigator-choice chemotherapy for ipilimumab-refractory melanoma (KEYNOTE-002): a randomised, controlled, phase 2 trial, Lancet Oncol. 16 (8) (2015) 908–918. [34] J.S. Weber, et al., Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 treatment (CheckMate 037): a randomised, controlled, open-label, phase 3 trial, Lancet Oncol. 16 (4) (2015) 375–384. [35] C. Robert, et al., Pembrolizumab versus ipilimumab in advanced melanoma, N. Engl. J. Med. 372 (26) (2015) 2521–2532.
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Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Synergistic inhibition of melanoma xenografts by Brequinar sodium and Doxorubicin Mathura Subangari Dorasamya,b, Aravind ABc, Kavitha Nellorec, Pooi-Fong Wonga,
T
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a
Department of Pharmacology, Faculty of Medicine, University of Malaya, Kuala Lumpur, 50603, Malaysia Aurigene Discovery Technologies, IPPP, University Malaya, Kuala Lumpur, 50603, Malaysia c Aurigene Discovery Technologies Limited, Electronic City, Bangalore, 560100, Karnataka, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: A375 Brequinar Doxorubicin In vivo Cyclin B1 pcdc-2 p21
Malignant melanoma continues to be a fatal disease for which novel and long-term curative breakthroughs are desired. One such innovative idea would be to assess combination therapeutic treatments – by way of combining two potentially effective and very different therapy. Previously, we have shown that DHODH inhibitors, A771726 and Brequinar sodium (BQR) induced cell growth impairment in melanoma cells. Similar results were seen with DHODH RNA interference (shRNA). In the present study, we showed that combination of BQR with doxorubicin resulted in synergistic and additive cell growth inhibition in these cells. In addition, in vivo studies with this combination of drugs demonstrated an almost 90% tumor regression in nude mice bearing melanoma tumors. Cell cycle regulatory proteins, cyclin B1 and its binding partner pcdc-2 and p21 were significantly downregulated and upregulated respectively following the combined treatment. Given that we have observed synergistic effects with BQR and doxorubicin, both in vitro and in vivo, these drugs potentially represent a new combination in the targeted therapy of melanoma.
1. Introduction Malignant melanoma is a melanocyte neoplasm that usually takes place in the skin and occasionally harbors mutations in genes including BRAF [1,2]. Each year, the number of estimated new cases of malignant melanoma is 132,000 and approximately 48,000 patients die from malignant melanoma globally. When malignant melanoma is diagnosed at the early stage (stage 0/I), the patient’s 5-year survival rate is more than 90% after surgical excision. However, when early detection fails, it tends to invade deeply and metastasize to lymph nodes and other organs. Median overall survival rate for melanoma patients with metastasis is less than 1 year [3]. The treatment of metastatic melanoma is rapidly evolving. The potent and specific BRAF inhibitors vemurafenib and dabrafenib, as compared with cytotoxic chemotherapeutic drugs, have significantly improved response rates, along with progression-free and overall survival in patients with metastatic melanoma with BRAF V600E or V600K mutations [4,5]. However, acquired resistance to BRAF inhibitors frequently develops through reactivation of the mitogen-activated protein kinase (MAPK) pathway, resulting in a median progression-free survival of 6 to 8 months [5–8]. In addition, the use of BRAF inhibitors may result in the development of secondary skin tumors, originating from a paradoxical activation of the MAPK pathway
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in cells without a BRAF mutation [4,9–14]. Given that the majority of BRAF inhibitor resistance occurs through reactivation of MAPK, several potent, non-ATP competitive MEK inhibitors were developed (eg, trametinib and cobimetinib) [15] that show no off-target effects on other kinases and are currently being used in clinical trials. MEK inhibitors are able to target MAPK-dependent tumors and exhibit distinct efficacies against BRAF- and KRAS-mutant melanomas [16,17]. Studies reveal that BRAF inhibitor-resistant tumor cells are highly sensitive to MEK inhibition and demonstrate that targeted pharmacological MEK inhibition may be a highly effective therapeutic alternative in BRAF inhibitor-resistant melanoma [17]. In light of these findings, focus has turned to dual inhibition of BRAF and its downstream target, MEK [18]. The results of combination therapy of MEK inhibitors and BRAF inhibitors are promising. New clinical trials confirm improved PFS progression-free survival with concurrent inhibition of BRAF and MEK. A Phase III, randomized study, compared patients with BRAF-mutated melanomas treated with vemurafenib 960 mg twice daily versus vemurafenib plus cobimetinib 60 mg daily for 21 days followed by 7 days off. The group on combination therapy displayed prolonged median PFS (9.9 months vs 6.2 months). The latest Phase III clinical trial of dabrafenib (BRAF inhibitor) and trametinib versus monotherapy with vemurafenib showed increased median PFS (11.4 months vs 7.3 months;
Corresponding author. E-mail address: [email protected] (P.-F. Wong).
https://doi.org/10.1016/j.biopha.2018.11.010 Received 1 September 2018; Received in revised form 2 November 2018; Accepted 2 November 2018 0753-3322/ © 2018 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/).
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antagonistic by taking into account the entire shape of the growth inhibition curve [23]. The resulting combination index (CI) theorem of Chou-Talalay offers quantitative definition for additive effect (CI = 1), synergism (CI < 1), and antagonism (CI > 1) in drug combinations.
HR 0.56; 95% confidence interval [CI]: 0.46– 0.69; P = 0.001) and improved overall survival at 12 months with combination therapy (72% vs 65%; HR 0.69; 95% CI: 0.53–0.89; P = 0.005) [19]. Despite these clinical benefits, the emergence of resistance to BRAF inhibitors and eventually to MEK inhibitors restricts the therapeutic efficacy of these kinase inhibitors. In an attempt to overcome this resistance, more potential molecules targeting other aberrant signalling pathways are in clinical development [20,21]. We have previously shown that DHODH inhibitors such as A771726 (Leflunomide metabolite) and Brequinar sodium (BQR) expose their anti-proliferative effects on melanoma, myeloma and lymphoma cells. These inhibitors arrested cancer cells growth in S-phase, decreased cMyc and increased p21 protein expression [22]. Furthermore, DHODH suppression by shRNA affected cell proliferation, c-Myc and its target protein, p21 in melanoma cells. In this study, we sought to investigate the effect of combining BQR and doxorubicin in vitro and in human melanoma xenograft. The combination treatment further sensitized the in vitro and in vivo melanoma tumor growth.
2.5. In vivo toxicity assessment of BQR and doxorubicin MTD is defined as the highest tolerable dose which does not produce major life threatening toxicity for the study duration [24]. Toxicity assessment of BQR and doxorubicin were performed using CD-1 mice, which are commonly used in toxicology and cancer research [25–27]. The MTD doses in this study were selected based on previous findings of the efficacy of BQR and doxorubicin in CD-1 mice [43–46]. Single dose of BQR (20 mg/kg) and doxorubicin (1 mg/kg) as well as in combination: BQR + doxorubicin (10 mg/kg + 1 mg/kg), BQR + doxorubicin (20 mg/kg + 1 mg/kg) were tested. Toxicity study was conducted in 5 mice per group for 14 days. Drugs were administered to CD-1 mice orally or intravenously (lateral tail vein) and toxicity was evaluated based on clinical symptoms such as lethargy, loss of body weight, diarrhoea and behavioural changes. All animal experiments were performed in accordance with the animal care and use protocol (Aurigene/ IAEC/PCD/018 E-13/09-2014) approved by the Institutional Animal Care and Use Committee, Aurigene Discovery Technologies.
2. Materials and methods 2.1. Cells Human melanoma (A375) cell line was obtained from American Type Cell Collections. A375 cells were grown in DMEM (Sigma, USA). The medium was supplemented with 10% heat-inactivated fetal bovine serum (Sigma, USA), 100 units/mL penicillin and 100 μg/mL streptomycin (Gibco, USA). Cells were maintained in a 5% CO2 atmosphere at 37 °C.
2.6. Establishment of tumor xenograft Athymic nude mice, aged 5 to 6 weeks, and weighing approximately 18–20 g from Harlan laboratories (Hyderabad, India) were housed in individually ventilated cages and were fed ad libitum with reverse osmosis treated water and gamma irradiated rodent pellet. A375 xenografts were established by subcutaneous injection of 5 × 106 A375 cells suspended in 150 μl of 1:1 ratio of Hank’s balanced salt solution and extracellular matrix. The mixture was injected onto the right flank of the mice. The individual animal body weights were recorded daily before the administration of test items and the tumor volumes were measured thrice weekly. All animal experiments were performed in accordance with the animal care and use protocol (Aurigene/IAEC/ PCD/018 E-13/09-2014) approved by the Institutional Animal Care and Use Committee, Aurigene Discovery Technologies.
2.2. Drugs and antibodies A771726, BQR and PLX 4720 were pharmaceutical standard with a purity of 99.50%, 98.80% and 99.55%, respectively and these drugs were synthesized at Aurigene Discovery Technologies Limited, Bangalore, India. Doxorubicin was purchased from Celon Labs, India. Antibodies for cyclin B1, pcdc-2 and p21 and loading control, GAPDH were procured from Cell Signaling Technology, USA. Secondary antibody, anti-rabbit polyclonal antibody was secured from Sigma, USA. 2.3. Cell proliferation assay
2.7. Assessment of the effect of combined drug treatments in tumor xenograft
A375 cells were seeded in 96-well plates at 1000 cells per well. The DHODH inhibitors - A771726 and BQR were dissolved in Hybrimax DMSO (Sigma, USA) whereas doxorubicin was dissolved in sterile distilled water. The additive or synergy effect between BQR and doxorubicin was determined using a non-fixed ratio method in which fixed concentrations of doxorubicin (0.3, 0.15, 0.075, 0.00375, 0.001875 and 0.009375 μM) were added with increasing concentrations of BQR (30.0, 10.0, 3.33, 1.11, 0.37, 0.123, 0.041 and 0.014 μM) and incubated for 72 h. Each plate contained control wells for vehicle (0.5% DMSO), BQR alone and doxorubicin alone in triplicates. Cell viability was determined using Alamar blue assay. Cells were treated with 50 μl/well of 1 mg/ml resazurin (Sigma, USA) dissolved in PBS (Sigma, USA). The fluorescence reading was measured at Ex/Em of 531/595 nm. Cell viability was calculated as a percentage of fluorescence or absorbance measured in the treated wells relative to the DMSO control wells. Experiments were repeated three times and mean ± SE was calculated.
When the mean tumor volume reached 100–150 mm3 in size, animals were randomized based on tumor volumes into seven groups and allocated nine animals for each group. The treatment groups are as following: (i) Vehicle control (diluent control), (ii) BQR (10 mg/kg), (iii) BQR (20 mg/kg), (iv) Doxorubicin (1 mg/kg), (v) BQR + Doxorubicin (10 mg/kg + 1 mg/kg), (vi) BQR + Doxorubicin (20 mg/kg + 1 mg/kg) and (vii) PLX 4720 (Vemurafenib), a BRAF inhibitor (20 mg/kg). BQR was formulated in 0.5% CMC + 0.25% tween 20 and administered orally once every alternate day, Doxorubicin was formulated in 5% dextrose and administered intravenously once every alternate day. Whereas, PLX 4720 was formulated in 5% DMSO + 1% methyl cellulose and administered orally once per day. The formulations were prepared fresh and used immediately. Body weights were monitored every day and tumor volumes were measured thrice weekly. The tumor volumes were measured using the formula: [A (length) x B (width)2]/2 [28]. The experiment was stopped at day 14, and all mice were sacrificed. The percentage of tumor growth inhibition was calculated using the following formula:
2.4. Assessment of the effect of combined drug treatments in vitro The effect of combined drug treatment of BQR and doxorubicin were examined using the median effect analysis method described by Chou and Talalay [23]. Calculation of combination index (CI) was performed using Compusyn software (Chou-Talalay, ComboSyn) to determine whether a combination is synergistic, additive, or
[vehicle control TV (day n – day 1)] – [drug TV (day n – day 1)]/[vehicle control TV (day n – day 1)] x 100, where TV = tumor volume and n = day of treatment 30
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evaluated for their anti-tumor activity in A375 xenograft model in athymic nude mice. PLX 4720 (Vemurafenib), a BRAF inhibitor approved for the treatment of late-stage melanoma [29] was used as the positive control. After 14 days of treatment, the tumor volume of mice administered with vehicle was 1090.0 ± 134.7 mm3 (Fig. 3A). By contrast, the tumor volumes of mice -administered with 20 mg/kg BQR; 10 mg/kg BQR + 1 mg/kg doxorubicin; 20 mg/kg BQR + 1 mg/kg doxorubicin and PLX 4720 were significantly lower than those of the vehicle control animals with tumor volumes of 404.6 ± 105 mm3; 388.6 ± 55.6 mm3; 217.2 ± 52.1 mm3 and 287.8 ± 50.7 mm3, respectively (Fig. 3A). However, there were no significant differences in the tumor volume of animals administered with single agent in comparison to the combination drugs as well as for those animals given combination drugs in comparison with PLX 4720. This is likely because of the large variances in the tumor volume between groups. To examine further the effectiveness of the drug treatment, the percentage tumor growth inhibition (TGI) was calculated relative to the vehicle control group. At the end of 14 days treatment, mice treated with 10 mg/kg and 20 mg/kg BQR as a single agent reduced tumor growth by 52.7 ± 6.5% and 70.6 ± 10.5% respectively, whereas doxorubicin at 1 mg/kg single agent suppressed tumor growth by only 36.1 ± 4.7% relative to the vehicle control (Fig. 3B). The positive control drug, PLX 4270 resulted in tumor growth inhibition (TGI) of 82.7 ± 4.6% relative to the vehicle control. The percentage TGI with 10 mg/kg BQR + 1 mg/kg doxorubicin (72.2 ± 5.2%) was significantly higher than that of treatment with 10 mg/kg BQR alone (52.7 ± 6.5%). Similarly, the percentage TGI with 20 mg/kg BQR + 1 mg/kg doxorubicin (89.9 ± 4.5%) was significantly higher than that of the treatment with 20 mg/kg BQR alone (70.6 ± 10.5%). The combination of 10 mg/kg BQR with 1 mg/kg doxorubicin, however, yielded a TGI of 72.2 ± 5.2% only compared to the TGI of PLX 4270 (82.7 ± 4.6%). Nevertheless, the combination of 20 mg/kg BQR with 1 mg/kg doxorubicin (89.9 ± 4.5%) resulted in higher percentage TGI compared to PLX 4270 (82.7 ± 4.6%). Analysis with Compusyn showed that doxorubicin co-administered with BQR at higher dosage synergistically inhibited tumor growth (Fig. 3C). This combination not only inhibited tumor growth more effectively but also reduced vascularity in the tumor compared to vehicle control, BQR, doxorubicin and PLX 4720 alone, (Fig. 4A and B). The maximal body weight change of the treated mice was below 10% compared to the vehicle control treated mice, indicating a lack of cumulative toxicity during treatment (Fig. 5).
2.8. Tumor lysate protein extraction and immunoblotting Excised tumors were minced and suspended in tissue protein extraction reagent (Cell lysis buffer) (Cell Signaling Technology, USA) supplemented with protease and phosphatase inhibitor cocktails (Sigma, USA) and homogenized. The homogenized tissue was transferred to a pre-chilled microcentrifuge tube, incubated on ice for 10 min and then centrifuged at 15,000 x g for 20 min at 4 °C. The supernatant containing total cellular proteins was collected for immunoblotting analysis. Extracted protein was quantified with Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Equal amount of protein was subjected to a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (GE Healthcare, USA). The membrane was blocked with Tris-buffer saline with 0.1% Tween-20 (TBST) containing 5% dry milk (Cell Signaling, USA). After blocking, the membrane was probed with appropriate primary antibodies overnight at 4 °C. On the next day, the binding of the primary antibodies was detected with anti-rabbit polyclonal secondary antibody labelled with horseradish peroxidase. The blot was developed with ECL Western blotting detection system (Pierce, USA) and subsequently the membrane was exposed to X-ray film. The intensity of each protein band was measured with ImageJ. 2.9. Statistical analysis Statistical significance of the differences between control and treatment was analyzed with Student’s t-test and one-way ANOVA with Dunnett’s or Tukey’s Multiple Comparison Tests, where applicable using GraphPad Prism 6.0. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 were considered statistically significant. 3. Results 3.1. BQR in combination with doxorubicin enhanced melanoma inhibition A375 cells were incubated with BQR or A771726 in combination with doxorubicin for 72 h. Synergistic or additive effects were achieved in most combinations except for BQR with 0.009375 μM doxorubicin. Combination of BQR with doxorubicin (Fig. 1A and B) showed greater dose-dependent proliferation inhibition than BQR alone (approximately 56.25 ± 2.6% inhibition at 30 μM) and doxorubicin alone (approximately 83.25 ± 4.33% at 0.3 μM) in A375 cells. Significant proliferation inhibition was achieved with 30 μM BQR in combination with 0.075 μM; 0.15 μM and 0.3 μM doxorubicin, with the percentage of inhibition of approximately 78.0 ± 6.03%; 91.25 ± 4.87% and 97.25 ± 0.63%, respectively in comparison to treatment with BQR alone. Whereas, the percentages of inhibition for treatment with 0.3 μM doxorubicin in combination with 30 μM and 10 μM BQR were approximately 97.25 ± 0.63 and 96.75 ± 0.85%, respectively.
3.4. Combination of brequinar and doxorubicin treatment target cyclin B1, pcdc-2 and p21 in A375 xenografts Previously, we have shown that treatment with BQR primarily induced S-phase and G2/M cell cycle arrest and p21 protein upregulation in vitro. Here, we showed that single treatments of BQR downregulated cyclin B1 expression by approximately 1.9-fold when compared to the vehicle control group. This protein was further decreased with the combination of 10 or 20 mg/kg BQR + 1 mg/kg doxorubicin as illustrated in Fig. 6A. Notably, the combination treatments significantly suppressed the expression of cyclin B1 by 2-2.4-fold compared to the single treatment of 20 mg/kg BQR; by 10.9–12.2-fold compared to doxorubicin treatment alone and by 3.4-3.8-fold compared to vehicle control (Fig. 6A). Interestingly, treatment with doxorubicin and PLX 4720 alone did not reduce the expression of this protein. To understand further, the binding partner of cyclin B1, pcdc-2 expression was also examined. The data presented in Fig. 6B shows pcdc-2 expression was downregulated in all treatment groups except doxorubicin when compared to the vehicle control group. The expression of pcdc2 was markedly downregulated by 5-20-fold in the presence of 10 mg/kg BQR + 1 mg/kg doxorubicin as compared to its respective single agents and vehicle control. Similarly, this downregulated trend of pcdc2 expression was observed in the combination treatment of 20 mg/
3.2. BQR in combination with doxorubicin was well tolerated in mice In vivo toxicity assessment of the single and combination of BQR and doxorubicin drugs was performed in CD-1 mice for a period of 14 days and mice weight was recorded (Fig. 2A). The tested doses of BQR and doxorubicin were well tolerated and no treatment related clinical signs or mortality was observed. Non-treated mice demonstrated a weight gain of 8% at the end of day 14. Instead of weight loss, mice with single treatment displayed a weight gain between 4–4.6% whereas mice with combination treatment saw a weight gain of no more than 1.4% (Fig. 2B). 3.3. Brequinar sodium potentiates anticancer effect of doxorubicin in A375 xenografts The in vitro synergistic effect of BQR and doxorubicin was further 31
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Fig. 1. BQR in combination with doxorubicin synergistically exerts inhibition of A375 melanoma cells. A375 cells were treated with BQR or doxorubicin alone or in combination for 72 h. Inhibition of proliferation was measured by Alamar blue assay. A, Percentage of proliferation inhibition by BQR (30.0, 10.0, 3.33, 1.11, 0.37, 0.123, 0.041 and 0.014 μM) in combination with doxorubicin (0.3, 0.15, 0.075, 0.00375, 0.001875 and 0.009375 μM). B, Percentage of proliferation inhibition of doxorubicin (0.3, 0.15, 0.075, 0.00375, 0.001875 and 0.009375 μM) in combination with BQR (30.0, 10.0, 3.3, 1.1 and 0.37 μM). Values shown are mean ± SE of 4 independent experiments. **P < 0.01 and ****P < 0.0001 (One-way ANOVA, Dunnett’s Multiple Comparison Tests). Brequinar and doxorubicin are abbreviated as BQR and Doxo, respectively.
4. Discussion
kg BQR + 1 mg/kg doxorubicin, albeit insignificant. The expression of the cell cycle regulatory protein, p21 in all single except doxorubicin and combination treatments were decreased as compared to the vehicle control. Nevertheless, as hypothesized, the expression of this protein was significantly upregulated by 0.3–0.5-fold in both the combination treatments as compared to treatment with BQR alone (Fig. 6C). Intriguingly, this protein was not detected with PLX 4720 (Fig. 6C and D).
Acknowledged as one of the most aggressive skin cancers, melanoma has limited therapeutic options and its occurrence is steadily and rapidly increasing. While this is true, there has been a host of discoveries and applications of new targeted therapy agents that have brought about significant benefits. Nevertheless, resistance to chemotherapy as well as undesirable side effects remain as a monumental challenge in the clinical treatment of malignant melanoma [30]. Past studies using cell lines derived from metastatic lesions resulted in cells becoming resistant to both MEK and PI3K inhibitors when grown in 3D Fig. 2. Effect of drug treatment on CD1 mice. A, Body weight of CD-1 mice during drug treatment. B, The percentage of body weight gain after 14 days of treatment in CD-1 mice. Mice were weighed daily and the percentage of body weight gain was obtained by subtracting mice weight three days before treatment from the weight on day 14. Brequinar and doxorubicin are abbreviated as BQR and Doxo, respectively.
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Fig. 3. Combination of BQR and doxorubicin achieved greater tumor inhibition in A375 melanoma xenograft. A375 xenograft mice were treated with vehicle control, BQR (10 mg/kg or 20 mg/kg, oral, every alternate day), doxorubicin (1 mg/kg, i.v. every alternate day) and PLX 4720 (20 mg/kg, oral, daily). Tumor volumes were measured once every 3 days. A, Data show mean tumor volume (mm3) ± standard error mean (SEM, error bars) of 9 mice per group. B, Percentage of tumor growth inhibition (TGI) ± standard error and CI values. TGI were analysed with the Compusyn software to show additive/synergistic effects. CI = 0.80 indicates additive effects, whereas CI = 0.44 indicates synergism. *P < 0.05 and **P < 0.01 relative to vehicle control (One-way ANOVA, Dunnett’s Multiple Comparison Test). # P < 0.05 and ###P < 0.001 relative to the respective single agent (One-way ANOVA, Tukey’s Multiple Comparison Test). Brequinar and doxorubicin are abbreviated as BQR and Doxo, respectively.
dabrafenib lasts only 6–8 months before the disease relapses especially in patients with metastatic melanoma [38]. With that in mind, rational combination approaches are strongly preferred in order to improve the overall patient progression-free survival (PFS), overcome or delay the development of multi-drug resistance and reduce the incidents of side effects [39–42]. In the present study, the anti-tumor effect of BQR alone and in combination with doxorubicin, an inhibitor of topo-isomerase 2 [43] was investigated. Preclinical studies of the maximum tolerated dose of BQR in several animal species have been reported previously. For instance, a dose of 193 mg/m2 per day induced a 10% lethality in mice for a period of 5 days every 4 weeks. Furthermore, preclinical studies in dogs showed that they were only able to tolerate a maximum dose of 6 mg/m2 per day on the same schedule [44]. Today, phase I of clinical trials with anticancer drugs recommend dosages for humans to be one third of that (13) or 2 mg/m2. However, previous reports have stated that doses of 100 mg/m2 (i.v.) daily for 5 days every 4 weeks had been safely administered to patients with advanced cancer and minimal prior treatment [45]. Building on from this knowledge, 2 doses of BQR were selected for this study, 10 mg/kg and 20 mg/kg. Doxorubicin is a widely used antineoplastic agent for many cancer types. However, it is ineffective against melanoma cells due to the frequent development of resistance [46–48]. In addition to its role as a RNA-synthesis inhibitor, He et al. [49] reported that doxorubicin significantly reduced both protein and mRNA expression of DHODH in U1690 cells in a concentration-dependent manner. Further to this,
culture. Similar outcome was also observed in in vivo studies, where MEK inhibitor, AZD6244 led to the stabilisation of established human melanoma xenografts, but not tumor regression [31]. In other words, as the melanomas progress, there seem to be functional redundancy between the various signalling pathways in these cells [32]. Hence, targeting multiple signalling pathways simultaneously could be a better strategy to induce melanoma regression. We have previously shown that Brequinar (BQR) produces antiproliferative effect on melanoma cells by inducing cell cycle arrest via the DHODH-independent pathway that is associated with p21 up-regulation and c-Myc down-regulation in vitro. In addition, we have also demonstrated that exogenous uridine was able to completely reverse the anti-proliferative effect mediated by BQR at all concentrations [22]. These results strongly suggest that two mechanisms, DHODH-independent and DHODH-dependent may possibly be involved in the anti-proliferative effect of BQR on A375 cells. Therefore, targeting multiple inhibitory pathways may improve clinical efficacy. The FDA approved melanoma inhibitors such as pembrolizumab targeting the PD-1 (programmed cell death protein 1, ipilumab targeting the CTLA-4, cytotoxic T lymphocyte-associated antigen 4, MEK inhibitor, trametinib and BRAF inhibitors, vemurafenib and dabrafenib represent an important milestone in more effective treatment of advanced melanoma [4,19,33–36]. Nevertheless, it is clear that the clinical use of these single-agent therapies have limitations. For example, ipilimumab only showed 4.5% objective response rate when used alone in a Phase II clinical trial [37]. The efficacy of vemurafenib and 33
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Fig. 4. Combination of BQR and doxorubicin achieved greater therapeutic effect in melanoma xenograft in vivo. A, Representive images of the location of tumor xenografts in atymic nude mice and B, tumor regression at the end of the study. Brequinar and doxorubicin are abbreviated as BQR and Doxo, respectively.
with multiple drugs (combined treatment) has been actively discussed and embraced in recent years. Brown et al. demonstrated that a combination of leflunomide and doxorubicin sensitized xenograft tumors derived from MDA-MB-231 cell lines. Moreover, our in vivo studies used a dosage of doxorubicin (1 mg/kg) that is approximately 0.1 times the recommended human dose based on body surface area [52]. The dosing schedule in this study is fairly short in comparison to various other reports. It is one that has been designed to achieve a prolonged drug exposure in an outpatient clinical setting, potentially yielding superior antitumor results. To further elucidate the underlying mechanism of melanoma xenograft inhibition, we analysed the expression profiles of important cell cycle regulator proteins such as p21, cyclin B1 and p-cdc-2. Interestingly, a clear and significant downregulation was seen in the expression of cyclin B1 and p-cdc-2 in the combined treatment. Correspondingly, the cell-cycle regulatory protein, p21 was observed to be substantially upregulated in both the combined treatments. In the cell cycle of eukaryotes, activation of cyclin B1-cdk 1 (cyclin dependent kinase 1) also known as cdc-2 brings the onset of mitosis. Cyclin B1 is involved in the transition from G2 to M phase however it becomes unregulated in cancer cells where it is often found overexpressed. Overexpression of cyclin B1 thus leads to uncontrolled cell growth [53–59]. Alternatively, cdc-2 phosphorylation inhibits cell differentiation at the G2/M phase that leads to an arrest in the entry of eukaryotic cell into mitosis [60]. The suppression of cell proliferation by upregulation of p21 expression is a prominent mechanism activated by several chemotherapy drugs that cause genotoxic stress [61]. As determined in this study, the suppressed expression of cyclin B1 and pcdc-2 with concomitant increase in the expression of p21 protein especially in the combined treatment further reaffirms our earlier in vitro findings [22] which suggest that these inhibitors arrest cancer cell proliferation primarily at S-phase and G2/M phase. Nevertheless, our observation of a reduction in p21 expression in all treatments compared to vehicle control in general, but an increase with combination treatment, leads us to the possibility of the dual role of p21 in oncogenesis as described by Cmielova et al. [62]. They suggest that the expression of p21 in cancer
Fig. 5. Percentage of body weight change in A375 xenograft mouse model during the treatment period. Mice were weighed daily and their body weight was expressed as a percentage of body weight change. Brequinar and doxorubicin are abbreviated as BQR and Doxo, respectively.
various studies have also reported that doxorubicin in combination with inhibitors such as berberine (in mouse xenograft) [50] and benzodiazepinedione [51] demonstrated a better therapeutic effect than either single drug alone. Our findings show that BQR in combination with doxorubicin synergistically suppressed the growth of A375 cells both in vitro and in vivo within 14 days of prolonged exposure to these inhibitors without any noticeable toxic profiles. These results provide the first in vitro and in vivo evidence that BQR co-administered with doxorubicin can potentiate the RNA-synthesis inhibitor effect via the DHODH pyrimidine pathway and as such, may provide promising therapeutic benefit as a combination therapy for melanoma. The rationale of targeted therapy 34
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Fig. 6. Combined treatment with 20 mg/kg BQR and 1 mg/kg doxorubicin resulted in greater downregulation of cyclin B1 and pcdc-2 in A375 melanoma xenograft. A, B and C, shows quantification of cyclin B1, pcdc-2 and p21, respectively whereas D, displays representative immunoblots of 4 animals of all 7 groups of mice. All the treated and non-treated groups of A375 melanoma xenografts were sacrificed on day 14 and tumors were excised. Tumor powder was prepared with liquid nitrogen and the protein was digested with cell lysis buffer. Protein quantification was performed with BCA kit and Western blot analysis was carried out. Equal amount of protein lysates were used with the antibodies indicated. GAPDH served as protein loading control. Data represents standard error mean ± (SEM, error bars) of 9 mice per group *, P < 0.05, **P < 0.01 and ***P < 0.001.
Declarations of interest
cells depends on the extent of the DNA damage. Low-level DNA damage leads to high expression of p21 and it induces cell cycle arrest whereas extensive DNA damage decreases the amount of p21 and the cancer cells undergoes apoptosis. As such, the role of p21 is context dependent as it can act as pro-apoptotic or anti-apoptotic in different conditions [63]. With this in mind, these results suggest that the upregulation of p21 is a pivotal mechanism for the enhanced suppression of melanoma cells.
None. Acknowledgement I would like to thank all Aurigene Discovery Technologies staff for their guidance and support. References
5. Conclusion
[1] E.D. Deeks, Nivolumab: a review of its use in patients with malignant melanoma, Drugs 74 (11) (2014) 1233–1239. [2] R. Okura, et al., Expression of AID in malignant melanoma with BRAFV600E mutation, Exp. Dermatol. 23 (5) (2014) 347–348. [3] E. Mashima, et al., Nivolumab in the treatment of malignant melanoma: review of the literature, Oncol. Ther. 8 (2015) 2045. [4] P.B. Chapman, et al., Improved survival with vemurafenib in melanoma with BRAF V600E mutation, N. Engl. J. Med. 364 (26) (2011) 2507–2516. [5] A. Hauschild, et al., Dabrafenib in BRAF-mutated metastatic melanoma: a multicentre, open-label, phase 3 randomised controlled trial, Lancet 380 (9839) (2012) 358–365. [6] D.B. Solit, N. Rosen, Resistance to BRAF inhibition in melanomas, N. Engl. J. Med. 364 (8) (2011) 772–774. [7] H. Shi, et al., Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy, Cancer Discov. 4 (1) (2014) 80–93. [8] E.M. Van Allen, et al., The genetic landscape of clinical resistance to RAF inhibition in melanoma, ASCO Annual Meeting Proceedings, (2013). [9] K.T. Flaherty, et al., Inhibition of mutated, activated BRAF in metastatic melanoma,
Findings from the present study revealed combination treatment of BQR and doxorubicin resulted in significant inhibitory effect on the growth of melanoma cells both in vitro and in vivo. The favourable synergistic effect and low toxic profiles of this treatment combination in melanoma xenograft model suggests that this combination has high potentials as targeted therapy for melanoma and warrants future pharmacokinetics and resistance studies. Funding This work was supported by Aurigene Discovery Technologies, India and University of Malaya Postgraduate Research Fund (PPP) Grant Number: PG007-2014B. 35
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[36] C. Garbe, S. Abusaif, T.K. Eigentler, Vemurafenib, Recent Results Cancer Res. 201 (2014) 215–225. [37] E.M. Hersh, et al., A phase II multicenter study of ipilimumab with or without dacarbazine in chemotherapy-naive patients with advanced melanoma, Invest. New Drugs 29 (3) (2011) 489–498. [38] J.A. Sosman, et al., Survival in BRAF V600–mutant advanced melanoma treated with vemurafenib, N. Engl. J. Med. 366 (8) (2012) 707–714. [39] B. Al-Lazikani, P. Workman, Unpicking the combination lock for mutant BRAF and RAS melanomas, Cancer Discov. 3 (1) (2013) 14–19. [40] S. Hu-Lieskovan, et al., Combining targeted therapy with immunotherapy in BRAFmutant melanoma: promise and challenges, J. Clin. Oncol. 32 (21) (2014) 2248–2254. [41] C. Nijenhuis, et al., Is combination therapy the next step to overcome resistance and reduce toxicities in melanoma? Cancer Treat. Rev. 39 (4) (2013) 305–312. [42] M. Voskoboynik, H.-T. Arkenau, Combination therapies for the treatment of advanced melanoma: a review of current evidence, Biochem. Res. Int. 2014 (2014). [43] D.A. Gewirtz, A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin, Biochem. Pharmacol. 57 (7) (1999) 727–741. [44] D.A. Noe, et al., Phase I and pharmacokinetic study of Brequinar sodium (NSC 368390), Cancer Res. 50 (15) (1990) 4595–4599. [45] C.L. Arteaga, et al., Phase I clinical and pharmacokinetic trial of Brequinar sodium (DuP 785; NSC 368390), Cancer Res. 49 (16) (1989) 4648–4653. [46] M. Panneerselvam, R. Bredehorst, C.W. Vogel, Resistance of human melanoma cells against the cytotoxic and complement-enhancing activities of doxorubicin, Cancer Res. 47 (17) (1987) 4601–4607. [47] M.G. Smylie, et al., A phase II, open label, monotherapy study of liposomal doxorubicin in patients with metastatic malignant melanoma, Invest. New Drugs 25 (2) (2007) 155–159. [48] D.A. Vorobiof, et al., Phase II study of pegylated liposomal doxorubicin in patients with metastatic malignant melanoma failing standard chemotherapy treatment, Melanoma Res. 13 (2) (2003) 201–203. [49] T. He, et al., Inhibition of the mitochondrial pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase by doxorubicin and brequinar sensitizes cancer cells to TRAIL-induced apoptosis, Oncogene 33 (27) (2014) 3538–3549. [50] A. Mittal, S. Tabasum, R.P. Singh, Berberine in combination with doxorubicin suppresses growth of murine melanoma B16F10 cells in culture and xenograft, Phytomedicine 21 (3) (2014) 340–347. [51] H.K. Koblish, et al., Benzodiazepinedione inhibitors of the Hdm2: p53 complex suppress human tumor cell proliferation in vitro and sensitize tumors to doxorubicin in vivo, Mol. Cancer Ther. 5 (1) (2006) 160–169. [52] K.K. Brown, et al., Adaptive reprogramming of de novo pyrimidine synthesis is a metabolic vulnerability in triple-negative breast cancer, Cancer Discov. 7 (4) (2017) 391–399. [53] S.K. Banerjee, et al., Expression of cdc2 and cyclin B1 in Helicobacter pylori-associated gastric MALT and MALT lymphoma: relationship to cell death, proliferation, and transformation, Am. J. Pathol. 156 (1) (2000) 217–225. [54] K.A. Hassan, et al., Cyclin B1 overexpression and resistance to radiotherapy in head and neck squamous cell carcinoma, Cancer Res. 62 (22) (2002) 6414–6417. [55] H. Kawamoto, H. Koizumi, T. Uchikoshi, Expression of the G2-M checkpoint regulators cyclin B1 and cdc2 in nonmalignant and malignant human breast lesions: immunocytochemical and quantitative image analyses, Am. J. Pathol. 150 (1) (1997) 15–23. [56] P. Rudolph, et al., Differential prognostic impact of the cyclins E and B in premenopausal and postmenopausal women with lymph node-negative breast cancer, Int. J. Cancer 105 (5) (2003) 674–680. [57] J.C. Soria, et al., Overexpression of cyclin B1 in early-stage non-small cell lung cancer and its clinical implication, Cancer Res. 60 (15) (2000) 4000–4004. [58] A. Wang, et al., Overexpression of cyclin B1 in human colorectal cancers, J. Cancer Res. Clin. Oncol. 123 (2) (1997) 124–127. [59] M. Zhao, et al., Expression profiling of cyclin B1 and D1 in cervical carcinoma, Exp. Oncol. 28 (1) (2006) 44–48. [60] D.O. Morgan, Principles of CDK regulation, Nature 374 (6518) (1995) 131–134. [61] T. Abbas, A. Dutta, p21 in cancer: intricate networks and multiple activities, Nat. Rev. Cancer 9 (6) (2009) 400–414. [62] J. Cmielova, M. Rezacova, p21Cip1/Waf1 protein and its function based on a subcellular localization [corrected], J. Cell. Biochem. 112 (12) (2011) 3502–3506. [63] S. Liu, W.R. Bishop, M. Liu, Differential effects of cell cycle regulatory protein p21(WAF1/Cip1) on apoptosis and sensitivity to cancer chemotherapy, Drug Resist. Updat. 6 (4) (2003) 183–195.
N. Engl. J. Med. 363 (9) (2010) 809–819. [10] C. Robert, J.-P. Arnault, C. Mateus, RAF inhibition and induction of cutaneous squamous cell carcinoma, Curr. Opin. Oncol. 23 (2) (2011) 177–182. [11] J. Carnahan, et al., Selective and potent Raf inhibitors paradoxically stimulate normal cell proliferation and tumor growth, Mol. Cancer Ther. 9 (8) (2010) 2399–2410. [12] P.A. Oberholzer, et al., RAS mutations are associated with the development of cutaneous squamous cell tumors in patients treated with RAF inhibitors, J. Clin. Oncol. 30 (3) (2012) 316–321. [13] L. Boussemart, et al., Prospective study of cutaneous side-effects associated with the BRAF inhibitor vemurafenib: a study of 42 patients, Ann. Oncol. 24 (6) (2013) 1691–1697. [14] F. Su, et al., RAS mutations in cutaneous squamous-cell carcinomas in patients treated with BRAF inhibitors, N. Engl. J. Med. 366 (3) (2012) 207–215. [15] K.D. Rice, et al., Novel carboxamide-based allosteric MEK inhibitors: discovery and optimization efforts toward XL518 (GDC-0973), ACS Med. Chem. Lett. 3 (5) (2012) 416–421. [16] G. Hatzivassiliou, et al., Mechanism of MEK inhibition determines efficacy in mutant KRAS-versus BRAF-driven cancers, Nature 501 (7466) (2013) 232–236. [17] D.B. Solit, et al., BRAF mutation predicts sensitivity to MEK inhibition, Nature 439 (7074) (2006) 358–362. [18] K.A. Tran, et al., MEK inhibitors and their potential in the treatment of advanced melanoma: the advantages of combination therapy, Drug Des. Devel. Ther. 10 (2016) 43. [19] C. Robert, et al., Improved overall survival in melanoma with combined dabrafenib and trametinib, N. Engl. J. Med. 372 (1) (2015) 30–39. [20] M. Hao, et al., Advances in targeted therapy for unresectable melanoma: new drugs and combinations, Cancer Lett. 359 (1) (2015) 1–8. [21] A. Niezgoda, P. Niezgoda, R. Czajkowski, Novel approaches to treatment of advanced melanoma: a review on targeted therapy and immunotherapy, Biomed Res. Int. 2015 (2015). [22] M.S. Dorasamy, et al., Dihydroorotate dehydrogenase inhibitors target c-Myc and arrest melanoma, myeloma and lymphoma cells at S-phase, J. Cancer 8 (15) (2017) 3086–3098. [23] T.-C. Chou, P. Talalay, Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors, Adv. Enzyme Regul. 22 (1984) 27–55. [24] S. Robinson, et al., A European pharmaceutical company initiative challenging the regulatory requirement for acute toxicity studies in pharmaceutical drug development, Regul. Toxicol. Pharmacol. 50 (3) (2008) 345–352. [25] S. Cui, C. Chesson, R. Hope, Genetic variation within and between strains of outbred Swiss mice, Lab. Anim. 27 (2) (1993) 116–123. [26] R. Chia, et al., The origins and uses of mouse outbred stocks, Nat. Genet. 37 (11) (2005) 1181–1186. [27] M.C. Rice, S.J. O’Brien, Genetic variance of laboratory outbred Swiss mice, Nature 283 (5743) (1980) 157–161. [28] D.S. Martin, et al., Therapeutic utility of utilizing low doses of N-(phosphonacetyl)L-aspartic acid in combination with 5-fluorouracil: a murine study with clinical relevance, Cancer Res. 43 (5) (1983) 2317–2321. [29] J. Tsai, et al., Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity, Proc. Natl. Acad. Sci. U. S. A. 105 (8) (2008) 3041–3046. [30] S. Liu, et al., Genetic variants in the genes encoding rho GTPases and related regulators predict cutaneous melanoma-specific survival, Int. J. Cancer 141 (4) (2017) 721–730. [31] N.K. Haass, et al., The mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor AZD6244 (ARRY-142886) induces growth arrest in melanoma cells and tumor regression when combined with docetaxel, Clin. Cancer Res. 14 (1) (2008) 230–239. [32] K.S. Smalley, et al., Multiple signaling pathways must be targeted to overcome drug resistance in cell lines derived from melanoma metastases, Mol. Cancer Ther. 5 (5) (2006) 1136–1144. [33] A. Ribas, et al., Pembrolizumab versus investigator-choice chemotherapy for ipilimumab-refractory melanoma (KEYNOTE-002): a randomised, controlled, phase 2 trial, Lancet Oncol. 16 (8) (2015) 908–918. [34] J.S. Weber, et al., Nivolumab versus chemotherapy in patients with advanced melanoma who progressed after anti-CTLA-4 treatment (CheckMate 037): a randomised, controlled, open-label, phase 3 trial, Lancet Oncol. 16 (4) (2015) 375–384. [35] C. Robert, et al., Pembrolizumab versus ipilimumab in advanced melanoma, N. Engl. J. Med. 372 (26) (2015) 2521–2532.
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