|Ahead of print publication
Antiangiogenic activity of zinc and zinc-sorafenib combination using the chick chorioallantoic membrane assay: A descriptive study
Manu Kumar1, Girish Gulab Meshram2, Tripti Rastogi1, Sonal Sharma3, Rachna Gupta1, Seema Jain1, Anisha Prasad1, Vikas Galav4, SK Bhattacharya1
1 Department of Pharmacology, University College of Medical Sciences and Guru Teg Bahadur Hospital, New Delhi, India
2 Department of Pharmacology, Postgraduate Institute of Medical Education and Research and Dr. Ram Manohar Lohia Hospital, New Delhi, India
3 Department of Pathology, University College of Medical Sciences and Guru Teg Bahadur Hospital, New Delhi, India
4 Department of Central Animal House, University College of Medical Sciences and Guru Teg Bahadur Hospital, New Delhi, India
Girish Gulab Meshram,
Department of Pharmacology, Postgraduate Institute of Medical Education and Research and Dr. Ram Manohar Lohia Hospital, New Delhi - 110 001
Source of Support: None, Conflict of Interest: None
Aim: Zinc, a trace element, is known for downregulating several proangiogenic growth factors and cytokines. However, its antiangiogenic activity is not adequately studied. The present study was aimed to evaluate the possible antiangiogenic activity of zinc via the chick chorioallantoic membrane (CAM) assay. Furthermore, the antiangiogenic activity of the combination therapy of zinc with various doses of sorafenib, a tyrosine kinase inhibitor, was evaluated.
Materials and Methods: A pilot study was initially conducted so as to select suitable doses of zinc and sorafenib. The antiangiogenic activity after combining zinc 2.5 μg/embryo with sorafenib 1 and 2 μg/embryo was also evaluated. The antiangiogenic activity was quantified in terms of total length of blood vessels, number of junctions, number of branching points, and mean length of the blood vessels.
Results: Zinc 2.5 μg/embryo showed significant (P < 0.05) antiangiogenic activity, as compared to the control group. However, its effect was not comparable to that of sorafenib 2 μg/embryo. The combination of zinc 2.5 μg/embryo with sorafenib 2 μg/embryo did not show an additive/synergistic effect. The combination of zinc 2.5 μg/embryo with sorafenib 1 μg/embryo produced an antiangiogenic activity which was comparable (P > 0.05) to that of sorafenib 2 μg/embryo.
Conclusion: Zinc caused significant antiangiogenic activity in the CAM assay. The lack of addition/synergism in the zinc-sorafenib combination could have been due to the variability in the dose/ratio selection. Addition of zinc to sorafenib therapy could improve treatment tolerability, reduce cost of therapy, and reduce the emergence of drug resistance. Future mechanistic studies could identify the exact pharmacodynamics of zinc as an angiogenesis inhibitor.
Keywords: Antiangiogenic, chick chorioallantoic membrane assay, sorafenib, zinc
|How to cite this URL:|
Kumar M, Meshram GG, Rastogi T, Sharma S, Gupta R, Jain S, Prasad A, Galav V, Bhattacharya S K. Antiangiogenic activity of zinc and zinc-sorafenib combination using the chick chorioallantoic membrane assay: A descriptive study. J Can Res Ther [Epub ahead of print] [cited 2020 Jan 28]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=257462
| > Introduction|| |
Angiogenesis, the formation of new blood vessels from preexisting ones, is critical for the progression and metastasis of several solid vascular tumors such as renal cell, hepatocellular, and colorectal cancers. The angiogenesis inhibitors approved for cancers include bevacizumab, sunitinib, and sorafenib. Bevacizumab blocks the vascular endothelial growth factor (VEGF), one of the major growth factors for angiogenesis. Sunitinib and sorafenib inhibit the enzyme tyrosine kinase, which regulates several proangiogenic growth factor receptors. However, the clinical benefits of these agents have been limited to only an increase in progression-free survival. Furthermore, due to an increase in the emergence of resistance to these agents, there is an enormous interest in researchers to develop novel angiogenesis inhibitors with efficient therapeutics.
Despite a large body of evidence pointing toward the antiangiogenic activity of zinc, the potential of zinc, as an angiogenesis inhibitor, remains unexplored. First, depletion of zinc has been reported to increase the expression of proangiogenic cytokines such as VEGF, interleukin 6 (IL-6), and IL-8 in prostate cancer cells. Second, zinc reduced the expression of hypoxia-inducible factor 1-α (HIF-1α), the master transcription factor for hypoxia-induced angiogenesis, in tumor cells. Third, zinc also increases the expression of p53 in tumor cells leading to reduced expression of VEGF. Fourth, zinc also reduces the levels of homeodomain-interacting protein kinase-2 (HIPK-2) and glycogen synthase kinase-3 β (GSK-3 β), both players in the process of angiogenesis., With this backdrop in mind, the present study was aimed to evaluate the possible antiangiogenic activity of zinc using the chick chorioallantoic membrane (CAM) assay. The possibility of an additive effect after combining zinc with various doses of sorafenib, an approved angiogenesis inhibitor, was also evaluated.
| > Materials and Methods|| |
The study was conducted at University College of Medical Sciences and Guru Teg Bahadur Hospital (UCMS and GTBH) after approval of the ‘Institutional Animal Ethics committee of UCMS and GTBH’ (Approval no. IAEC/2014-106).
Pure zinc powder was purchased from Merck Ltd., Mumbai, India, and sorafenib was purchased from Sigma-Aldrich, St. Louis, MO, USA.
Chick chorioallantoic membrane assay
Fresh fertilized chicken eggs of the Lohmann Brown variety were purchased from Keggfarms, Gurgaon, India, and disinfected with 70% ethanol. The eggs were then placed in an incubator (United National Scientific Co., Kolkata, India) at 37°C and 65% relative humidity for embryogenesis. On the 3rd day of incubation, a tiny window was made on the apex of each of the eggs and the eggs were sealed with paraffin film (American Can Co., Greenwich, CT, USA) to avoid contamination of the egg contents. On the 4th day of incubation, the windows on the eggs were reopened and the test/control drugs were applied on the developing CAMs before resealing the eggs. On the 14th day of incubation, the CAMs were separated for quantifying the angiogenesis [Figure 1].
|Figure 1: Photographs of the pilot study showing a 14th day fertilized egg subjected to normal saline (a), zinc 2.5 μg/embryo (b), sorafenib 2 μg/embryo (c), and zinc 10 μg/embryo (d)|
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Quantification of angiogenesis
The CAMs were photographed (Model ProgRes C5, Jenoptik Inc, Jena, Germany) at 4X. After identifying and specifying the regions of interest in the images, the images were optimized, thresholded, and skeletonized so as to calculate the (1) total length, (2) number of junctions, (3) number of branches, and (4) mean branch length of the blood vessels, using the software Angiosys version 2.0 (Cellworks, Caltag Medsystems Ltd., Buckingham, UK) [Figure 2].
|Figure 2: Final processed images by Angiosys 2.0 showing angiogenesis in the chick chorioallantoic membranes subjected to normal saline (a), zinc 2.5 μg/embryo (b), sorafenib 2 μg/embryo (c), zinc 2.5 μg/embryo + sorafenib 2 μg/embryo (d), and zinc 2.5 μg/embryo + sorafenib 1 μg/embryo (e)|
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Doses of zinc and sorafenib
For determining suitable doses of zinc and sorafenib, a pilot study was initially conducted by administering zinc at doses of 2.5, 5, 10, and 20 μg/embryo and sorafenib at doses of 2 and 3 μg/embryo to the CAMs (n = 6 in each group)., Normal saline was used as the control. Subsequently, the doses of zinc and sorafenib showing maximal efficacy in the CAM assay, among the doses evaluated, were combined together so as to observe the effect of the combination therapy. Subsequently, a lower dose of sorafenib, i.e., 1 μg/embryo was combined with the most efficacious dose of zinc to evaluate their combined efficacy.
Results are expressed as mean ± standard error of mean. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by multiple Tukey's comparison test. P < 0.05 was considered statistically significant.
Two-way multivariate ANOVA (MANOVA) was utilized to compare the main effect of sorafenib and zinc and the interaction effect between sorafenib and zinc on the total length, branches, junctions, and mean branch length of blood vessels. For the two-way MANOVA, sorafenib included two levels (0 and 2 μg/embryo) and zinc contained two levels (0 and 2.5 μg/embryo).
| > Results|| |
Zinc at the dose of 2.5 μg/embryo showed statistically significant (P < 0.05) antiangiogenic activity, as compared to the control group. As two of the CAMs in the zinc 5 μg/embryo group did not show any embryogenesis, the effect of the zinc 5 μg/embryo group could not be compared to that of the zinc 2.5 μg/embryo group. The 10 and 20 μg/embryo doses of zinc showed toxic effects leading to no embryogenesis in all of the CAMs [Figure 1]. Sorafenib 2 and 3 μg/embryo showed statistically significant (P < 0.05) antiangiogenic activity, as compared to the control group [Table 1]. However, sorafenib 2 μg/embryo was chosen as the reference standard owing to a lower risk of toxicity to the CAMs. The antiangiogenic activity of zinc 2.5 μg/embryo was significantly (P < 0.05) lower than that of sorafenib 2 μg/embryo. The results of the pilot study indicated zinc 2.5 μg/embryo and sorafenib 2 μg/embryo as efficacious and nontoxic doses, and hence were selected for the subsequent part of the study [Figure 2], [Figure 3] and [Table 1].
|Table 1: Antiangiogenic activity of zinc, sorafenib, and their combinations in the chick chorioallantoic membrane assay|
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|Figure 3: Histograms showing the antiangiogenic activity of zinc, sorafenib, and their combinations in the chick chorioallantoic membrane assay C, control; Z 2.5, zinc 2.5 μg/embryo; Z 5, zinc 5 μg/embryo; S 1, sorafenib 1 μg/embryo; S 2, sorafenib 2 μg/embryo; and S 3, sorafenib 3 μg/embryo|
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The combination of zinc 2.5 μg/embryo with sorafenib 2 μg/embryo showed statistically significant (P < 0.05) antiangiogenic activity, as compared to the control group. However, its effect was not significantly (P < 0.05) superior to that of sorafenib 2 μg/embryo. Zinc 2.5 μg/embryo combined with sorafenib 1 μg/embryo showed statistically significant (P < 0.05) antiangiogenic activity, as compared to the control group. Furthermore, this combination did not show a statistically significant (P < 0.05) difference from the antiangiogenic activity of the sorafenib 2 μg/embryo group [Table 1].
Zinc 2.5 μg/embryo significantly (P < 0.05) reduced three of the parameters observed for quantifying angiogenesis, i.e., total length of the blood vessels, number of branching points, and number of junctions, but not the mean branch length (P = 0.89) of the blood vessels, as compared to the control group. Sorafenib and both the combination therapy groups significantly (P < 0.05) reduced all the four abovementioned quantification parameters, as compared to the control group [Table 1] and [Figure 2].
Following two-way MANOVA, the main effect for sorafenib yielded F ratios of F (1, 20) = 421.79, F (1, 20) = 347.93, F (1, 20) = 445.64, and F (1, 20) = 8.24 for total length, branches, junctions, and mean branch length, respectively, with P < 0.05, indicating a significant difference between sorafenib 0 μg/embryo and sorafenib 2 μg/embryo. The main effect for zinc yielded F ratios of F (1, 20) = 72.34, F (1, 20) = 50.81, F (1, 20) = 66.81, and F (1, 20) = 4.50 for total length, branches, junctions, and mean branch length, respectively, with P < 0.05, indicating that the effect was significantly different between zinc 0 μg/embryo and zinc 2.5 μg/embryo. There was a statistically significant interaction between sorafenib and zinc on total length (F [1, 20] = 41.01, P < 0.05), branches (F [1, 20] = 26.98, P < 0.05), and junctions (F (1, 20) = 40.71, P < 0.05) but not on the mean branch length (F [1, 20] = 1.26, P = 0.28). The interaction effect between sorafenib and zinc on each of the dependent variables is depicted graphically in [Figure 4].
|Figure 4: Plots showing the means of total length, branches, junctions, and mean branch length for each combination of groups of sorafenib (0 and 2 μg/embryo) and zinc (0 and 2.5 μ/embryo). Parallel lines indicate no interaction effect. Converging lines indicate a positive interaction effect. Diverging lines indicate a negative interaction effect. S 0, sorafenib 0 μg/embryo; S 2, sorafenib 2 μg/embryo; Z 0, zinc 0 μg/embryo; and Z 2.5, zinc 2.5 μg/embryo|
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| > Discussion|| |
In recent years, numerous reports have focused on the anticancer activity of zinc. However, no study so far has evaluated the antiangiogenic activity of zinc in the CAM assay. In the present study, zinc was found to be effective in inhibiting angiogenesis in the CAM assay. Furthermore, the combination of zinc with a lower dose of sorafenib, i.e., 1 μg/embryo was found to be as effective as a higher dose of sorafenib, i.e., 2 μg/embryo in inhibiting angiogenesis. These findings will carve the way for future studies, evaluating the role of zinc in angiogenesis.
The CAM assay is a classic model for screening novel antiangiogenic agents. It provides a natural in vivo environment for angiogenesis with an advantage of direct application of test compounds to the membrane, the effect of which can be visualized and quantified in real time.
To ensure appropriate doses of zinc and sorafenib, in the present study, a pilot study was initially conducted. For the pilot study, the doses of zinc 2.5, 5, 10, and 20 μg/embryo and sorafenib 2 and 3 μg/embryo were selected on the basis of previous studies.,
Zinc has shown to have a conflicting role in the process of angiogenesis. On the one hand, zinc fingers, zinc sulfate, and zinc oxide nanoparticles have been reported to promote angiogenesis by upregulating proangiogenic factors such as VEGF and fibroblast growth factor and downregulating matrix metalloproteinases; while on the other hand, zinc has also been reported to reduce proangiogenic factors such as VEGF, IL-2, IL-6, IL-8, HIF-1α, HIPK-2, and GSK-3 β and increase the expression of p53, an antiangiogenic factor, in previous reports.,,,,,,,,, Zinc also inhibits the inhibitor of κB kinase (IκB kinase) which results in nuclear factor κB inhibition downstream of the mitogen-activated protein kinase pathway leading to reduced angiogenesis. Furthermore, the binding of zinc to endostatin, a potent angiogenesis inhibitor, is essential for the antiangiogenic activity of endostatin. The overall action of zinc appears to be antiangiogenic, as found in the present study, which is in alignment with the results of previous studies., The differential effect of zinc on angiogenesis could be dependent on the particle size, particle surface chemistry, dose dilution, cell type, and mode of delivery. Sorafenib, an approved angiogenesis inhibitor, acts by inhibiting the enzyme tyrosine kinase, responsible for the activation of VEGF and PGDF receptors.
The use of angiogenesis inhibitors in cancer therapy has resulted in moderate improvement in survival rates along with considerable adverse effects. The major roadblocks identified for their limited success include patient/tumor heterogeneity, limited dose schedules due to drug toxicity, and emergence of drug resistance. To tackle the tangible hurdles of drug resistance and drug toxicity, a combination of two different angiogenesis inhibitors acting via diverse mechanisms, or a combination of one angiogenesis inhibitor with one low-dose anticancer agent, is often sought. Zinc could be an excellent option for such a combination therapy due to its dual action of inhibiting both angiogenesis and tumor cell growth and also due to its involvement in several angiostatic signaling pathways. However, in the present study, the combination of zinc 2.5 μg/embryo with sorafenib 2 μg/embryo showed only a marginal improvement in the antiangiogenic activity, as compared to sorafenib 2 μg/embryo monotherapy, making the interaction effect partially enhancing in nature. The lack of synergism or additive effect between sorafenib and zinc could be explained by the fact that the phenomena of synergism and addition between two angiostatic agents are dose/ratio dependent. A small change in the dose/ratio of even one of the agents may lead to loss of synergism/addition., Furthermore, in anticancer therapy, the sequence of administering two agents can influence the efficacy of the combination therapy. The presence of a drug–drug interaction between sorafenib and zinc, partially buffering the overall antiangiogenic activity of the zinc–sorafenib combination, cannot be ruled out either. When a lower dose of sorafenib, i.e., 1 μg/embryo was combined with zinc 2.5 μg/embryo, the effect was comparable to that of sorafenib 2 μg/embryo monotherapy. Hence, combining zinc with sorafenib could lower the dose of sorafenib, making the treatment more tolerable and cost-effective. Furthermore, GSK-3 activity induced by sorafenib in cancer cells is one of the potential causes for its resistance. Zinc inhibits GSK-3 and could be an effective option as a combination agent with sorafenib. However, more in-depth studies are warranted to ascertain these findings.
The present study has numerous limitations. First, the doses of zinc and sorafenib were not calculated by lethal dose50 estimation by dose-dependent toxicity studies, keeping in mind the narrow therapeutic window of metallic ions in therapeutics. Second, although zinc showed significant antiangiogenic activity in the CAM assay, due to the absence of mechanistic studies, measuring levels of proangiogenic and antiangiogenic factors affected by zinc, the exact pharmacodynamics of zinc, as an antiangiogenic agent, remains yet unexplained. Third, only one model, i.e., the CAM assay was employed to measure the antiangiogenic activity of zinc. Fourth, although there was a significant interaction effect found between zinc and sorafenib in three of the dependent variables, pharmacokinetic/molecular/isobolographic studies, evaluating the nature and molecular mechanism underlying this interaction effect, were not conducted. Future studies employing a combination of assays for accurate identification of the cellular and molecular events in angiogenesis targeted by zinc could give a complete picture of its antiangiogenic potential. Future isobolographic studies could identify the exact nature of the interaction between zinc and sorafenib.
| > Conclusion|| |
The study shows that zinc effectively inhibits angiogenesis in the CAM assay. The mechanism postulated for its antiangiogenic action includes inhibition of multiple proangiogenic growth factors and cytokines. The combination of zinc with other angiogenesis inhibitors, including sorafenib, could provide an excellent opportunity for improving angiostatic therapy by reducing the incidence of drug resistance, reducing cost of therapy, and improving treatment tolerability in patients. Future mechanistic studies could shed light on the exact pharmacodynamics and role of zinc in angiogenesis for its successful translation to the clinic, as an effective angiogenesis inhibitor.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| > References|| |
Vasudev NS, Reynolds AR. Anti-angiogenic therapy for cancer: Current progress, unresolved questions and future directions. Angiogenesis 2014;17:471-94.
Chabner BA, Barnes J, Neal J, Olson E, Mujagic H, Sequist L, et al
. Targeted therapies: Tyrosine kinase inhibitors, monoclonal antibodies, and cytokines. In: Brunton LL, editor. Goodman and Gillman's Pharmacological Basis of Therapeutics. New York, USA: McGraw-Hill; 2011. p. 1731-53.
Ebos JM, Kerbel RS. Antiangiogenic therapy: Impact on invasion, disease progression, and metastasis. Nat Rev Clin Oncol 2011;8:210-21.
van Beijnum JR, Nowak-Sliwinska P, Huijbers EJ, Thijssen VL, Griffioen AW. The great escape; the hallmarks of resistance to antiangiogenic therapy. Pharmacol Rev 2015;67:441-61.
Golovine K, Uzzo RG, Makhov P, Crispen PL, Kunkle D, Kolenko VM, et al.
Depletion of intracellular zinc increases expression of tumorigenic cytokines VEGF, IL-6 and IL-8 in prostate cancer cells via NF-kappaB-dependent pathway. Prostate 2008;68:1443-9.
Nardinocchi L, Pantisano V, Puca R, Porru M, Aiello A, Grasselli A, et al.
Zinc downregulates HIF-1α and inhibits its activity in tumor cells in vitro
and in vivo
. PLoS One 2010;5:e15048.
Dhawan DK, Chadha VD. Zinc: A promising agent in dietary chemoprevention of cancer. Indian J Med Res 2010;132:676-82.
] [Full text]
Margalit O, Simon AJ, Yakubov E, Puca R, Yosepovich A, Avivi C, et al.
Zinc supplementation augments in vivo
antitumor effect of chemotherapy by restoring p53 function. Int J Cancer 2012;131:E562-8.
Ilouz R, Kaidanovich O, Gurwitz D, Eldar-Finkelman H. Inhibition of glycogen synthase kinase-3beta by bivalent zinc ions: Insight into the insulin-mimetic action of zinc. Biochem Biophys Res Commun 2002;295:102-6.
Ribatti D, Vacca A, Roncali L, Dammacco F. The chick embryo chorioallantoic membrane as a model for in vivo
research on anti-angiogenesis. Curr Pharm Biotechnol 2000;1:73-82.
Khoo CP, Micklem K, Watt SM. A comparison of methods for quantifying angiogenesis in the matrigel assay in vitro
. Tissue Eng Part C Methods 2011;17:895-906.
Uzzo RG, Crispen PL, Golovine K, Makhov P, Horwitz EM, Kolenko VM, et al.
Diverse effects of zinc on NF-kappaB and AP-1 transcription factors: Implications for prostate cancer progression. Carcinogenesis 2006;27:1980-90.
Nowak-Sliwinska P, Weiss A, van Beijnum JR, Wong TJ, Ballini JP, Lovisa B, et al.
Angiostatic kinase inhibitors to sustain photodynamic angio-occlusion. J Cell Mol Med 2012;16:1553-62.
Bozym RA, Chimienti F, Giblin LJ, Gross GW, Korichneva I, Li Y, et al.
Free zinc ions outside a narrow concentration range are toxic to a variety of cells in vitro
. Exp Biol Med (Maywood) 2010;235:741-50.
Barui AK, Veeriah V, Mukherjee S, Manna J, Patel AK, Patra S, et al.
Zinc oxide nanoflowers make new blood vessels. Nanoscale 2012;4:7861-9.
Kaji T, Fujiwara Y, Yamamoto C, Sakamoto M, Kozuka H. Stimulation by zinc of cultured vascular endothelial cell proliferation: Possible involvement of endogenous basic fibroblast growth factor. Life Sci 1994;55:1781-7.
Pasqualini R, Barbas CF 3rd
, Arap W. Vessel maneuvers: Zinc fingers promote angiogenesis. Nat Med 2002;8:1353-4.
Prasad AS. Effects of zinc deficiency on th1 and th2 cytokine shifts. J Infect Dis 2000;182 Suppl 1:S62-8.
Saghiri MA, Asatourian A, Orangi J, Sorenson CM, Sheibani N. Functional role of inorganic trace elements in angiogenesis-part II: Cr, si, zn, cu, and S. Crit Rev Oncol Hematol 2015;96:143-55.
Boehm T, O'reilly MS, Keough K, Shiloach J, Shapiro R, Folkman J, et al.
Zinc-binding of endostatin is essential for its antiangiogenic activity. Biochem Biophys Res Commun 1998;252:190-4.
Nishida N, Yano H, Nishida T, Kamura T, Kojiro M. Angiogenesis in cancer. Vasc Health Risk Manag 2006;2:213-9.
Tada-Oikawa S, Ichihara G, Suzuki Y, Izuoka K, Wu W, Yamada Y, et al.
Zn(II) released from zinc oxide nano/micro particles suppresses vasculogenesis in human endothelial colony-forming cells. Toxicol Rep 2015;2:692-701.
Osterhoudt KC, Penning TM. Drug toxicity and poisoning. In: Brunton LL, editor. Goodman and Gillman's Pharmacological Basis of Therapeutics. New York, USA: McGraw-Hill; 2011. p. 73-87.
Albiges L, Choueiri T, Escudier B, Galsky M, George D, Hofmann F, et al.
Asystematic review of sequencing and combinations of systemic therapy in metastatic renal cancer. Eur Urol 2015;67:100-10.
Kawazoe H, Bilim VN, Ugolkov AV, Yuuki K, Naito S, Nagaoka A, et al.
GSK-3 inhibition in vitro
and in vivo
enhances antitumor effect of sorafenib in renal cell carcinoma (RCC). Biochem Biophys Res Commun 2012;423:490-5.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]