Flavones and Flavonols may have Clinical Potential as CK2 Inhibitors in Cancer Therapy

Mark F. McCarty*

Corresponding Author

Mark F. McCarty

Catalytic Longevity, 7831 Rush Rose Drive Apt. 316, Carlsbad, California, 92009, USA; Tel. 760-216-7272; E-mail: markfmccarty@gmail.com

Affiliation

Mark F. McCarty*

Catalytic Longevity, 7831 Rush Rose Drive, Apt. 316, Carlsbad, California 92009, USA

Corresponding Author

Mark F. McCarty

Catalytic Longevity, 7831 Rush Rose Drive Apt. 316, Carlsbad, California, 92009, USA; Tel. 760-216-7272; E-mail: markfmccarty@gmail.com

Article History

Received: January 21st, 2015; Accepted: March 3rd, 2015; Published: March 4th, 2015

Cite this Article

McCarty MF. Flavones and flavonols may have clinical potential as CK2 inhibitors in cancer therapy. Cancer Stud Mol Med Open J. 2015; 2(1): 39- 51. doi: 10.17140/CSMMOJ-2-105

Copyright

©2015 McCarty MF. This is an open access article distributed under the Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Doi

10.17140/CSMMOJ-2-105

The serine-threonine kinase CK2, which targets over 300 cellular proteins, is over expressed in all cancers, presumably reflecting its ability to promote proliferation, spread, and survival through a wide range of complementary mechanisms. Via an activating phosphorylation of Cdc373, a co-chaperone which partners with Hsp90, CK2 prolongs the half-life of protein kinases that promote proliferation and survival in many cancers, including Akt, Src, EGFR, Raf-1, and several cyclin-dependent kinases. CK2 works in other ways to boost the activity of signaling pathways that promote cancer aggressiveness and chemoresistance, including those driven by Akt, NF-kappaB, hypoxia-inducible factor-1, beta-catenin, STAT3, hedgehog, Notch1, and the androgen receptor; it also promotes the epidermal-mesenchymal transition and aids efficiency of DNA repair. Several potent and relatively specific inhibitors of CK2 are now being evaluated as potential cancer drugs; CX-4945 has shown impressive activity in cell culture studies and xenograft models, and is now entering clinical trials. Moreover, it has long been recognized that the natural flavone apigenin can inhibit CK2, with a Ki near 1 micromolar; more recent work indicates that a range of flavones and flavonols, characterized by a planar structure and hydroxylations at the 7 and 4’ positions - including apigenin, luteolin, keampferol, fisetin, quercetin, and myricetin - can inhibit CK2 with Ki s in the sub-micromolar range. This finding is particularly intriguing in light of the numerous studies demonstrating that each of these agents can inhibit the growth of cancer cells lines in vitro and of human xenografts in nude mice. These studies attribute the cancer-retardant efficacy of flavones/flavonols to impacts on a bewildering array of cellular targets, including those whose activities are boosted by CK2; it is reasonable to suspect that, at least in physiologically achievable concentrations, these agents may be achieving these effects primarily via CK2 inhibition. Inefficient absorption and rapid conjugation limit the bioefficacy of orally administered flavonoids; however, the increased extracellular beta-glucuronidase of many tumors may give tumors privileged access to glucuronidated flavonoids, and nanopartical technology can improve the bioavailability of these agents. Enzymatically modified isoquercitrin has particular promise as a delivery vehicle for quercetin. Hence, it may be worthwhile to explore the clinical potential of flavones/flavonols as CK2 inhibitors for cancer therapy.

CK2 IS OVER-EXPRESSED AND UP-REGULATES PROLIFERATION, SPREAD, AND SURVIVAL IN CANCER

CK2, a serine-threonine kinase once known as casein kinase 2, is a ubiquitously expressed tetramer comprised of two catalytic subunits - α and/or α’ - and two regulatory β subunits that direct it to specific targets. CK2 is capable of phosphorylating a huge range of cellular proteins; over 300 physiological targets have been documented to date (though ironically casein is not one of them!).1Its level of expression and its sub-cellular localization determine its activity, as post-translational modifications or allosteric interactions are thought to have little impact in that regard; moreover, no gain-of-function mutants of this kinase are known.

Virtually all cancer cell lines studied to date overexpress CK2 protein, relative to its expression in normal tissues of origin; moreover, they tend to route a higher proportion of this protein to the cell nucleus.2 This is not likely to be accidental, as high CK2 activity works in a bewildering number of complementary ways to promote cellular proliferation and spread, while suppressing apoptosis. Hence, cancer cells which overexpress CK2 will tend to be selected for.

CK2 MODULATES A PLETHORA OF SIGNALING PATHWAYS

One of CK2’s most intriguing and ramified effects is to phosphorylate, and thereby activate, the co-chaperone Cdc37.3,4 Activated Cdc37 interacts with Hsp90 to provide chaperoning activity for a broad range of protein kinases, many of which play a role in promoting cell proliferation and survival. These include Akt, Src, EGFR, PDGFR, Raf-1, IKK, RIP1, Cdc2, Cdk2, Cdk4, and Cdk6. This chaperoning activity tends to slow the proteolytic degradation of these kinases, prolonging their effective half-lives; this activity is particularly crucial for the survival of certain mutant constitutively active forms of these kinases often found in cancers. To date, CK2 is the only upstream kinase known to confer activation on Cdc37 - for which reason assessment of Cdc37 phosphorylation at Ser13 has been proposed as a strategy for determining CK2 activity in vivo.5

But CK2 works in a number of additional ways to boost the activity of signaling pathways that make cancer more aggressive and harder to kill:

Akt

While CK2 boosts Akt expression via Hsp90-cdc37- mediated stabilization, it can also work in various complementary ways to increase the phosphorylation and activation of this key kinase, which promotes cellular proliferation while acting in a number of ways to inhibit apoptosis. CK2 phosphorylates Akt directly at Ser129; this up-regulates the activation of Akt mediated by PDK1 and mTORC2, and facilitates its association with Hsp90.6,7 And CK2 inhibits phosphatase activities that target Akt; it phosphorylates and thereby reduces the activity of the crucial cancer suppressor PTEN, and also promotes proteasomal degradation of PML, a protein which is an obligate component of a nuclear complex that dephosphorylates Akt within the nucleus.8,9,10,11 CK2 also has the potential to work upstream from Akt, enhancing its activation by up-regulating certain tyrosine kinase signaling pathways.

NF-kappaB

Numerous studies show that CK2 inhibition suppresses NF-kappaB activity in cancer cell lines, whereas overexpression of this kinase boosts NF-kappaB activity.12-27 CK2 promotes degradation of IkappaB; this can reflect an activating phosphorylation of IKKbeta, as well as a direct phosphorylation of IkappaB that renders it more sensitive to proteolytic cleavage by calpain.13,14,19,24 CK2 activity also has been reported to somehow boost the expression of IKK-i/IKKepsilon, an alternative IkappaB kinase complex capable of promoting IkappaB degradation.17 And the transcriptional activity of p65 is enhanced by a phosphorylation of Ser529 conferred by CK2.20

Hypoxia-inducible factor-1 (HIF-1)

CK2 enhances the transcriptional activity of HIF-1, even though it doesn’t increase the protein expression or nuclear binding of this factor.28,29,30 Some evidence suggests that this reflects a reduction of p53 levels; nuclear p53 somehow antagonizes the transcriptional activity of HIF-1.29CK2’s impact on p53 level, in turn, may reflect phosphorylations of MDM2 that enhance its ability to promote proteasomal degradation of p53.

Beta-Catenin

Many studies show that CK2 inhibition decreases Wntbeta-catenin signaling.22,31-39 Down-regulation of Akt, which stabilizes beta-catenin through inhibition of glycogen synthase kinase-3 and also via a direct phosphorylation on Ser552, evidently can contribute to this effect.37,38 However, CK2 also phosphorylates beta-catenin directly on Thr393, an effect which likewise prolongs the half-life and promotes the transcriptional activity of this factor.32,33

STAT3

There are several reports that inhibition of CK2 suppresses STAT3 phosphorylation and activation in cancer cell lines.40,41,42 The basis of this effect is not yet clear. In some cell lines, suppression of IL-6 expression may contribute to this effect.

Hedgehog

In human lung cancer cells, CK2 activity has been shown to boost the mRNA and protein expression of Gli1, and to enhance the half-life and transcriptional activity of this key mediator of hedgehog signaling.43 CK2 can directly phosphorylate Gli1, and it has been suggested that this may be responsible for the positive impact of CK2 on hedgehog signaling.

Notch1

In human lung cancer cell lines expressing Notch1, inhibition of CK2 activity suppresses Notch1-driven transcription, whereas forced overexpression of CK2 has the opposite effect.44 This may reflect the fact that CK2 activity increases the half-life of Notch1 protein.

Androgen Receptor

CK2 inhibitors suppress androgen receptor-mediated transcription in prostate cancer cell lines, at least in part by blocking androgen-induced nuclear translocation of the receptor.45,46,47 The direct target of CK2 in this effect has not been identified.

DNA Repair

CK2-mediated phosphorylations of XRCC1 and MDC1, nuclear proteins which play a key role in the repair of DNA single-strand and double-strand breaks, respectively, are required for their proper activity.48-53 Hence, inhibition of CK2 can boost the killing activity of DNA-damaging cytotoxins not only by up-regulating mechanisms of apoptosis, but also by impeding the efficiency of DNA repair.

Epidermal-Mesenchymal Transition

Studies with CK2 inhibitors demonstrate that CK2 activity can promote the epidermal-mesenchymal transition necessary for invasive behavior by boosting expression of vimentin, snail, and smad2/3, while suppressing that of E-cadherin.54,55,56,57,58For some reason this effect is most prominent in cancer cells which overexpress CK2 alpha catalytic subunits, relative to CK2 beta regulatory subunits.59,60

NEW DRUGS FOR INHIBITION OF CK2 - CX-4945

These considerations make it abundantly clear that well tolerated and effective pharmaceutical inhibitors of CK2 may have a bright future in oncology - both as agents for slowing cancer growth and spread, and as adjuvants to chemo- or radiotherapy. Some pharmaceutical companies are moving aggressively to evaluate the potential of this approach, and the highly potent and orally active CK2 inhibitor CX-4945 has shown impressive anti-cancer activity in mouse xenograft models, in doses which the animals appear to tolerate well.61,62 Moreover, in doses that don’t greatly retard tumor growth, CX-4945 considerably amplifies response of an ovarian cancer xenograft to gemcitabine and cisplatin - though the somewhat greater weight loss in the mice receiving combination therapy suggests that toxicity might also be increased to a degree.63 This agent is now entering clinical trials, and its progress should be followed with the greatest interest.

FLAVONES/FLAVONOLS AS NATURAL INHIBITORS OF CK2

However, there are other known inhibitors of CK2, one being the dietary flavone apigenin. Indeed, long before the development of the more potent pharmaceutical inhibitors of CK2, this agent was employed as a relatively specific inhibitor of CK2 in cell culture studies, with a Ki near 1 µM.64 There are indeed a number of studies, both in cancer cell culture and in mouse xenograft models, showing that apigenin can exert cancer-retardant and chemo-potentiating effects. In xenograft models, apigenin has shown activity whether administered parenterally or orally, alone or as an adjuvant to chemotherapy.65-81 Intriguingly, many of the effects of apigenin on signaling pathways reported in cell culture or xenograft studies are parallel to those of CK2 inhibition, including down-regulated activity of Akt,4,74,82,83,84,85,86 HIF1,65,67,87-91 NF-kappaB,4,17,21,28,92,93,94,95 STAT3,4,94,96 beta-catenin,97,98 Gli1,99 AR,100,101 and Cdc37,4 and up-regulated p53.94,102-109 Indeed, Zhao and colleagues have recently proposed that inhibition of CK2 is a key mediator of apigenin’s anti-cancer activity in multiple myeloma cells.4 A survey of the burgeoning cancer research literature involving apigenin - 476 citations on Pubmed at present - reveals apigenin can influence a truly dizzying array of molecular targets in cancer cells; it is reasonable to suspect that, rather than directly inhibiting dozens of separate targets, it must be influencing one or more signaling factors that have a remarkably broad impact on the molecular biology of cancer cells. CK2 may be the crucial target in this regard. However, none of the studies in which apigenin has been administered in cancer-retardant doses to xenograft-bearing mice have assessed the impact of apigenin on tumor CK2 activity. A study assessing this - perhaps by measuring Ser13 phosphorylation of Cdc37 in tumors - would be worthwhile; and it would also be intriguing to see whether apigenin administration has any significant additional impact on cancer growth in animals that are already receiving potent doses of CX-4945; if CK2 is apigenin’s key target, little additional benefit might be seen.

Although apigenin is considered the prototype flavone inhibitor of CK2, recent studies show that other naturally-occuring flavones and flavonols have similar or slightly more potent inhibitory activity. Working in vitro with human recombinant CK2, Lolli and colleagues have recently reported that apigenin, luteolin, kaempferol, fisetin, quercetin, and myricetin can inhibit CK2 with Ki s of 0.8, 0.5, 0.4, 0.35, 0.55, and 0.92 µM, respectively.110 This inhibition is competitive with respect to the phosphodonor substrate ATP. All effective compounds are planar and are hydroxylated at the7 and 4’ positions. Hydroxylations at 5, 3, and 3’ positions do not greatly add to or detract from activity.

These findings may help to explain the curious fact that every one of these flavones or flavonols has been reported to exert anti-cancer effects, both in cancer cell cultures, and in xenografted mice. Here are citations for the xenograft studies: apigenin,65-81 luteolin,111-122 kaempferol,123 fisetin,124,125,126 quercetin,127-145 myrcetin.146There are at least 53 published studies in which flavones or flavonols have decreased the growth of human xenografts in nude mice.

It seems likely that, ultimately, a drug such as CX-4945 will offer the most convenient and effective way to address the CK2 activity of clinical cancer. However, this or comparable drugs will not be available for several years, and when available will initially only be approved for use in a limited number of cancers - and will doubtless be staggeringly expensive to use for off-label purposes. For this reason, it would be prudent to give serious attention to the possibility that apigenin or related flavones/flavonols might be clinically useful for suppressing CK2 activity in some sufficiently high dosage schedule. This might be assessed by pharmacokinetic studies in which a marker for CK2 activity, such as phosphorylation of Cdc37 - or Thr145 phosphorylation of p21, employed as a marker in studies with CX-454961 - is determined in leukocytes or some other accessible cell type. The efficacy of a given agent will presumably reflect it absorbability, the rapidity with which it is conjugated once absorbed (glucuronidation or sulfation), and its capacity to pass through cell walls. Pharmaceutical innovations which optimize absorbability might make this approach more feasible.141 With respect to quercetin, the approved food additive enzymatically-modified isoquercitrin (EMIQ), unlike quercetin, is highly soluble, but is metabolized to yield free quercetin at the intestinal brush border; a human pharmacokinetic study found that when equimolar amounts of quercetin and EMIQ were administered orally, the plasma levels of quercetin achieved were twenty-fold higher with EMIQ.147-151This agent apparently has not yet been tested in rodent tumor models.

Rapid conjugation of absorbed flavonoids limits their capacity to exert intracellular effects.152 It is therefore fortunate that some tumors may have privileged access to flavone/ flavonol glucuronide conjugates, owing to the fact that extracellular beta-glucuronidase activity tends to be elevated in tumors, particularly in their hypoxic/necrotic regions.153,154 Infiltrating immune cells may be the chief source of this activity. Moreover, the tendency of extracellular pH to be acidic in such regions can be expected to amplify their beta-glucuronidase activity.155,156,157 Many investigators have proposed or presented evidence that glucuronide-masked anti-cancer agents - including flavonoids - can be selectively activated within tumors.154,157-165 Hence, the rapid glucuronidation of flavones and flavonols may not be an insuperable obstacle to the capacity of these compounds to inhibit CK2 in vivo. Perhaps this mechanism contributes to the demonstrable efficacy of flavones/flavonols in mouse xenograft studies; co-administration of a beta-glucuronidase inhibitor might clarify this. A corollary of this consideration, however, is that measurement of CK2 activity in healthy tissues following oral administration of flavones/flavonols may underestimate the capacity of these agents to inhibit CK2 within tumors.

JOINT INHIBITION OF HDAC6 AND CK2 TO TARGET HSP90 FUNCTION

In light of the fact that inhibition of the chaperoning function of Hsp90-Cdc37 plays a key role in the cancer-retardant efficacy of CK2 inhibitors, it is pertinent to note that acetylation of Hsp90 notably reduces its chaperoning activity.166-170 The cytosolic deacetylase HDAC6 targets these acetylations of Hsp90, restoring its activity. Hence, type II histone deacetylase inhibitors have the potential to complement the impact of CK2 inhibitors on the chaperoning of many pro-oncogenic kinases. Moreover, it has recently emerged that sulforaphane can function as an inhibitor of HDAC6 within cells;171 this phenomenon may be clinically relevant, as acute ingestion of 68 g of broccoli sprouts has been reported to suppress global histone deacetylase activity in peripheral blood mononuclear cells.166 This finding may be of particular interest, in light of the fact that HDAC6, rather like CK2, works in multifarious ways to sustain malignant cellular behavior, and is emerging as a key target for cancer therapy.172,173 It would be of interest to determine whether flavones/flavonols and sulforaphane might complement each other’s efficacy in integrative cancer therapy.

A POTENTIAL COUNTERVAILING EFFECT - NRF2 ACTIVATION

Although the great majority of reports examining quercetin’s impact on cancer, in vitro or in vivo, with or without concurrent chemotherapy, conclude that quercetin has cancer suppressive activity, one recent study found that, in low micromolar concentrations, quercetin protected a human ovarian cancer cell line from a range of cytotoxic drugs; concurrent quercetin administration decreased the cancer-retardant efficacy of cisplatin in a xenograft model. This effect was traced to quercetin’s ability to activate nrf2 and thereby increase the expression of antioxidant enzymes, glutathione, and glutathione-dependent detoxicant enzymes. The ability of phase 2 induction via nfr2 activation to promote chemoresistance in some cancers has been demonstrated. Hence, while a number of studies describe a chemosensitizing effect for quercetin in cancer models135,136, 174,175,176 including a report that low concentrations of quercetin sensitize some ovarian cancer cell lines to cisplatin - the possibility remains that quercetin (and presumably other phase 2-inductive flavonols) may promote chemoresistance in some cancers. (The “flip side” of this observation is that quercetin has potential for protecting healthy tissues from chemotherapy drugs, as demonstrated in mice.177-181 These considerations, in any case, do not speak to quercetin’s potential utility as an adjuvant for slowing cancer growth.

EVALUATING THE HYPOTHESIS

As noted above, EMIQ may be the most appropriate agent to study in pre-clinical and clinical trials, owing to its ability to promote absorption of quercetin. In cancer xenograft models, the impact of EMIQ administration on Ser13 phosphorylation of Cdc37 in the tumor could be determined to assess this agent’s ability to suppress CK2 activity in vivo. Positive results in such studies could then encourage clinical cancer trials with EMIQ. Rather than expecting objective response, it would be more realistic to hope that flavonol administration will slow the growth and spread of cancer, as it does in rodent models. A placebo-controlled design might thus be required to establish clinical efficacy. The extent of Ser13 phosphorylation of Cdc37 in leukocytes could be measured as a surrogate for CK2 inhibition in the cancer - bearing in mind, however, that quercetin metabolites might have greater activity within inflamed tumor tissue.

In regard to toxicity considerations, it should be noted that knockout of the alpha subunits of CK2 results in embryonic lethality.182,183 However, flavonols in vivo would achieve at best only partial inhibition of CK2. In rodent studies with CX-4945, cancer control is noted with doses that are not overtly toxic to the animals. Phase I clinical trials with this agent have not yet been reported, so it is not clear what the dose-limiting toxicities of CK2 inhibitors will be. Flavonols are of course prominent phytochemicals in natural diets. The toxicological evaluation of EMIQ in rodents has been described by Valentova and colleagues; 147 when fed at up to 2.5% of diet to rats for 13 weeks, yellowish discoloration of bones and urine was noted, and weight gain was slightly decreased at the highest doses. At 5% of diet, isoquercetin feeding to male rats was associated with significant declines in body weight, hemoglobin, triglycerides, bilirubin, and phosphorus, with small increases in the relative weights of the lungs and testes.184 EMIQ has been accorded GRAS status for use as a food additive. These considerations suggest that it would be reasonably safe to test EMIQ in doses of several grams daily in Phase I cancer trials.

1. Meggio F, Pinna LA. One-thousand-and-one substrates of protein kinase CK2? FASEB J. 2003; 17(3): 349-368. doi: 10.1096/ fj.02-0473rev

2. Tawfic S, Yu S, Wang H, Faust R, Davis A, Ahmed K. Protein kinase CK2 signal in neoplasia. Histol Histopathol. 2001; 16(2): 573-582.doi: 10.14670/HH-16.573

3. Miyata Y, Nishida E. CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37. Mol Cell Biol. 2004; 24(9): 4065-4074. doi: 10.1128/ MCB.24.9.4065-4074.2004

4. Zhao M, Ma J, Zhu HY, et al. Apigenin inhibits proliferation and induces apoptosis in human multiple myeloma cells through targeting the trinity of CK2, Cdc37 and Hsp90. Mol Cancer. 2011; 10: 104. doi: 10.1186/1476-4598-10-104

5. Miyata Y, Nishida E. Evaluating CK2 activity with the antibody specific for the CK2-phosphorylated form of a kinase-targeting cochaperone Cdc37. Mol Cell Biochem. 2008; 316(1-2): 127-134. doi: 10.1007/s11010-008-9818-1

6. Di MG, Brustolon F, Pinna LA, Ruzzene M. Dephosphorylation and inactivation of Akt/PKB is counteracted by protein kinase CK2 in HEK 293T cells. Cell Mol Life Sci. 2009; 66(20): 3363-3373. doi: 10.1007/s00018-009-0108-1

7. Siddiqui-Jain A, Drygin D, Streiner N, et al. CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy. Cancer Res. 2010; 70(24): 10288-10298. doi: 10.1158/0008-5472.CAN-10-1893

8. Shehata M, Schnabl S, Demirtas D, et al. Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia. Blood. 2010; 116(14): 2513-2521. doi: 10.1182/blood-2009-10-248054

9. Barata JT. The impact of PTEN regulation by CK2 on PI3K-dependent signaling and leukemia cell survival. Adv Enzyme Regul. 2011; 51(1): 37-49. doi: 10.1016/j.advenzreg.2010.09.012

10. Kang NI, Yoon HY, Kim HA, et al. Protein kinase CK2/ PTEN pathway plays a key role in platelet-activating factor-mediated murine anaphylactic shock. J Immunol. 2011 1; 186(11): 6625-6632. doi: 10.4049/jimmunol.1100007

11. Chatterjee A, Chatterjee U, Ghosh MK. Activation of protein kinase CK2 attenuates FOXO3a functioning in a PML-dependent manner: implications in human prostate cancer. Cell Death Dis. 2013; 4: e543. doi: 10.1038/cddis.2013.63

12. Romieu-Mourez R, Landesman-Bollag E, Seldin DC, Traish AM, Mercurio F, Sonenshein GE. Roles of IKK kinases and protein kinase CK2 in activation of nuclear factor-kappaB in breast cancer. Cancer Res. 2001; 61(9): 3810-3818.

13. Shen J, Channavajhala P, Seldin DC, Sonenshein GE. Phosphorylation by the protein kinase CK2 promotes calpain-mediated degradation of IkappaBalpha. J Immunol. 2001; 167(9): 4919-4925. doi: 10.4049/jimmunol.167.9.4919

14. Romieu-Mourez R, Landesman-Bollag E, Seldin DC, Sonenshein GE. Protein kinase CK2 promotes aberrant activation of nuclear factor-kappaB, transformed phenotype, and survival of breast cancer cells. Cancer Res. 2002; 62(22): 6770-6778.

15. Kato T, Jr., Delhase M, Hoffmann A, Karin M. CK2 Is a CTerminal IkappaB Kinase Responsible for NF-kappaB Activation during the UV Response. Mol Cell. 2003; 12(4): 829-839.doi: 10.1016/s10972765(03)00358-7

16. Cavin LG, Romieu-Mourez R, Panta GR, et al. Inhibition of CK2 activity by TGF-beta1 promotes IkappaB-alpha protein stabilization and apoptosis of immortalized hepatocytes. Hepatology. 2003; 38(6): 1540-1551. doi: 10.1016/j.hep.2003.09.019

17. Eddy SF, Guo S, Demicco EG, et al. Inducible IkappaB kinase/IkappaB kinase epsilon expression is induced by CK2 and promotes aberrant nuclear factor-kappaB activation in breast cancer cells. Cancer Res. 2005; 65(24): 11375-11383. doi: 10.1158/0008-5472.CAN-05-1602

18. Piazza FA, Ruzzene M, Gurrieri C, et al. Multiple myeloma cell survival relies on high activity of protein kinase CK2. Blood. 2006; 108(5): 1698-1707. doi: 10.1182/blood-2005-11-013672

19. Yu M, Yeh J, Van WC. Protein kinase casein kinase 2 mediates inhibitor-kappaB kinase and aberrant nuclear factorkappaB activation by serum factor(sin head and neck squamous carcinoma cells. Cancer Res. 2006; 66(13): 6722-6731. doi: 10.1158/0008-5472.CAN-05-3758

20. Parhar K, Morse J, Salh B. The role of protein kinase CK2 in intestinal epithelial cell inflammatory signaling. Int J Colorectal Dis. 2007; 22(6): 601-609. doi: 10.1007/s00384-006-0193-7

21. Hamacher R, Saur D, Fritsch R, Reichert M, Schmid RM, Schneider G. Casein kinase II inhibition induces apoptosis in pancreatic cancer cells. Oncol Rep. 2007; 18(3): 695-701. doi: 10.3892/or.18.3.695

22. Dominguez I, Sonenshein GE, Seldin DC. Protein kinase CK2 in health and disease: CK2 and its role in Wnt and NFkappaB signaling: linking development and cancer. Cell Mol Life Sci. 2009; 66(11-12): 1850-1857. doi: 10.1007/s00018-009- 9153-z

23. Brown MS, Diallo OT, Hu M, et al. CK2 modulation of NFkappaB, TP53, and the malignant phenotype in head and neck cancer by anti-CK2 oligonucleotides in vitro or in vivo via sub50-nm nanocapsules. Clin Cancer Res. 2010; 16(8): 2295-2307. doi: 10.1158/1078-0432.CCR-09-3200

24. Tsuchiya Y, Asano T, Nakayama K, Kato T, Jr., Karin M, Kamata H. Nuclear IKKbeta is an adaptor protein for IkappaBalpha ubiquitination and degradation in UV-induced NF-kappaB activation. Mol Cell. 2010; 39(4): 570-582. doi: 10.1016/j.molcel.2010.07.030

25. Trembley JH, Unger GM, Tobolt DK, et al. Systemic administration of antisense oligonucleotides simultaneously targeting CK2alpha and alpha’ subunits reduces orthotopic xenograft prostate tumors in mice. Mol Cell Biochem. 2011; 356(1-2): 21- 35. doi: 10.1007/s11010-011-0943-x

26. Trembley JH, Unger GM, Korman VL, et al. Nanoencapsulated anti-CK2 small molecule drug or siRNA specifically targets malignant cancer but not benign cells. Cancer Lett. 2012; 315(1): 48-58. doi: 10.1016/j.canlet.2011.10.007

27. Zheng Y, McFarland BC, Drygin D, et al. Targeting Protein Kinase CK2 Suppresses Pro-survival Signaling Pathways and Growth of Glioblastoma. Clin Cancer Res. 2013; 19(23): 6484- 6494. doi: 10.1158/1078-0432.CCR-13-0265

28. Mottet D, Ruys SP, Demazy C, Raes M, Michiels C. Role for casein kinase 2 in the regulation of HIF-1 activity. Int J Cancer. 2005; 117(5): 764-774. doi: 10.1002/ijc.21268

29. Hubert A, Paris S, Piret JP, Ninane N, Raes M, Michiels C. Casein kinase 2 inhibition decreases hypoxia-inducible factor-1 activity under hypoxia through elevated p53 protein level. J Cell Sci. 2006; 119(Pt 16): 3351-3362. doi: 10.1242/jcs.03069

30. Ampofo E, Kietzmann T, Zimmer A, Jakupovic M, Montenarh M, Gotz C. Phosphorylation of the von Hippel-Lindau protein (VHL by protein kinase CK2 reduces its protein stability and affects p53 and HIF-1alpha mediated transcription. Int J Biochem Cell Biol. 2010; 42(10): 1729-1735. doi: 10.1016/j. biocel.2010.07.008

31. Song DH, Sussman DJ, Seldin DC. Endogenous protein kinase CK2 participates in Wnt signaling in mammary epithelial cells. J Biol Chem. 2000; 275(31): 23790-23797. doi: 10.1074/ jbc.M909107199

32. Song DH, Dominguez I, Mizuno J, Kaut M, Mohr SC, Seldin DC. CK2 phosphorylation of the armadillo repeat region of betacatenin potentiates Wnt signaling. J Biol Chem. 2003; 278(26): 24018-24025. doi: 10.1074/jbc.M212260200

33. Seldin DC, Landesman-Bollag E, Farago M, Currier N, Lou D, Dominguez I. CK2 as a positive regulator of Wnt signalling and tumourigenesis. Mol Cell Biochem. 2005; 274(1-2): 63-67. doi: 10.1007/s11010-005-3078-0

34. Tapia JC, Torres VA, Rodriguez DA, Leyton L, Quest AF. Casein kinase 2 (CK2increases survivin expression via enhanced beta-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription. Proc Natl Acad Sci U S A. 2006; 103(41): 15079-15084. doi: 10.1073/pnas.0606845103

35. Wang S, Jones KA. CK2 controls the recruitment of Wnt regulators to target genes in vivo. Curr Biol. 2006; 16(22): 2239- 2244. doi: 10.1016/j.cub.2006.09.034

36. Lee AK, Ahn SG, Yoon JH, Kim SA. Sox4 stimulates sscatenin activity through induction of CK2. Oncol Rep. 2011; 25(2): 559-565. doi: 10.3892/or.2010.1091

37. Ponce DP, Maturana JL, Cabello P, et al. Phosphorylation of AKT/PKB by CK2 is necessary for the AKT-dependent upregulation of beta-catenin transcriptional activity. J Cell Physiol. 2011; 226(7): 1953-1959. doi: 10.1002/jcp.22527

38. Ponce DP, Yefi R, Cabello P, et al. CK2 functionally interacts with AKT/PKB to promote the beta-catenin-dependent expression of survivin and enhance cell survival. Mol Cell Biochem. 2011; 356(1-2): 127-132. doi: 10.1007/s11010-011-0965-4

39. Kim J, Hwan KS. CK2 Inhibitor CX-4945 Blocks TGFbeta1-Induced Epithelial-to-Mesenchymal Transition in A549 Human Lung Adenocarcinoma Cells. PLoS ONE. 2013; 8(9): e74342.. doi: 10.1371/journal.pone.0074342

40. Piazza FA, Ruzzene M, Gurrieri C, et al. Multiple myeloma cell survival relies on high activity of protein kinase CK2. Blood. 2006; 108(5): 1698-1707. doi: 10.1182/blood-2005-11-013672

41. Lin YC, Hung MS, Lin CK, et al. CK2 inhibitors enhance the radiosensitivity of human non-small cell lung cancer cells through inhibition of stat3 activation. Cancer Biother Radiopharm. 2011; 26(3): 381-388. doi: 10.1089/cbr.2010.0917

42. Zhao M, Ma J, Zhu HY, et al. Apigenin inhibits proliferation and induces apoptosis in human multiple myeloma cells through targeting the trinity of CK2, Cdc37 and Hsp90. Mol Cancer. 2011; 10: 104. doi: 10.1186/1476-4598-10-104

43. Zhang S, Wang Y, Mao JH, et al. Inhibition of CK2alpha down-regulates Hedgehog/Gli signaling leading to a reduction of a stem-like side population in human lung cancer cells. PLoS ONE. 2012; 7(6): e38996. doi: 10.1111/jcmm.12068

44. Zhang S, Long H, Yang YL, et al. Inhibition of CK2alpha down-regulates Notch1 signalling in lung cancer cells. J Cell Mol Med. 2013; 17(7): 854-862. doi: 10.1111/jcmm.12068

45. Ryu BJ, Baek SH, Kim J, et al. Anti-androgen receptor activity of apoptotic CK2 inhibitor CX4945 in human prostate cancer LNCap cells. Bioorg Med Chem Lett. 2012; 22(17): 5470-5474. doi: 10.1016/j.bmcl.2012.07.031

46. Yao K, Youn H, Gao X, et al. Casein kinase 2 inhibition attenuates androgen receptor function and cell proliferation in prostate cancer cells. Prostate. 2012; 72(13): 1423-1430. doi: 10.1002/pros.22493

47. Gotz C, Bachmann C, Montenarh M. Inhibition of protein kinase CK2 leads to a modulation of androgen receptor dependent transcription in prostate cancer cells. Prostate. 2007; 67(2): 125-134. doi: 10.1002/pros.20471

48. Loizou JI, El-Khamisy SF, Zlatanou A, et al. The protein kinase CK2 facilitates repair of chromosomal DNA single-strand breaks. Cell. 2004; 117(1): 17-28. doi: 10.1016/s0092-8674- (04)00206-5

49. Parsons JL, Dianova II, Finch D, et al. XRCC1 phosphorylation by CK2 is required for its stability and efficient DNA repair. DNA Repair (Amst). 2010; 9(7): 835-841. doi: 10.1016/j.dnarep.2010.04.008

50. Strom CE, Mortusewicz O, Finch D, et al. CK2 phosphorylation of XRCC1 facilitates dissociation from DNA and single-strand break formation during base excision repair. DNA Repair (Amst). 2011; 10(9): 961-969. doi: 10.1016/j. dnarep.2011.07.004

51. Siddiqui-Jain A, Bliesath J, Macalino D, et al. CK2 inhibitor CX-4945 suppresses DNA repair response triggered by DNAtargeted anticancer drugs and augments efficacy: mechanistic rationale for drug combination therapy. Mol Cancer Ther. 2012; 11(4): 994-1005. doi: 10.1158/1535-7163.MCT-11-0613

52. Chapman JR, Jackson SP. Phospho-dependent interactions between NBS1 and MDC1 mediate chromatin retention of the MRN complex at sites of DNA damage. EMBO Rep. 2008; 9(8): 795-801. doi: 10.1038/embor.2008.103

53. Becherel OJ, Jakob B, Cherry AL, et al. CK2 phosphorylation-dependent interaction between aprataxin and MDC1 in the DNA damage response. Nucleic Acids Res. 2010; 38(5): 1489- 1503. doi: 10.1093/nar/gkp1149

54. Belguise K, Guo S, Yang S, et al. Green tea polyphenols reverse cooperation between c-Rel and CK2 that induces the aryl hydrocarbon receptor, slug, and an invasive phenotype. Cancer Res. 2007; 67(24): 11742-11750. doi: 10.1158/0008-5472.CAN07-2730

55. MacPherson MR, Molina P, Souchelnytskyi S, et al. Phosphorylation of serine 11 and serine 92 as new positive regulators of human Snail1 function: potential involvement of casein kinase-2 and the cAMP-activated kinase protein kinase A. Mol Biol Cell. 2010; 21(2): 244-253. doi: 10.1091/mbc.E09-06-0504

56. Su YW, Xie TX, Sano D, Myers JN. IL-6 stabilizes Twist and enhances tumor cell motility in head and neck cancer cells through activation of casein kinase 2. PLoS ONE. 2011; 6(4): e19412. doi: 10.1371/journal.pone.0019412

57. Zou J, Luo H, Zeng Q, Dong Z, Wu D, Liu L. Protein kinase CK2alpha is overexpressed in colorectal cancer and modulates cell proliferation and invasion via regulating EMT-related genes. J Transl Med. 2011; 9: 97. doi: 10.1186/1479-5876-9-97

58. Kim J, Hwan KS. CK2 Inhibitor CX-4945 Blocks TGFbeta1-Induced Epithelial-to-Mesenchymal Transition in A549 Human Lung Adenocarcinoma Cells. PLoS ONE. 2013; 8(9): e74342. doi: 10.1371/journal.pone.0074342

59. Deshiere A, Duchemin-Pelletier E, Spreux E, et al. Regulation of epithelial to mesenchymal transition: CK2beta on stage. Mol Cell Biochem. 2011; 356(1-2): 11-20. doi: 10.1007/s11010- 011-0942-y

60. Deshiere A, Duchemin-Pelletier E, Spreux E, et al. Unbalanced expression of CK2 kinase subunits is sufficient to drive epithelial-to-mesenchymal transition by Snail1 induction. Oncogene. 2013; 32(11): 1373-1383. doi: 10.1038/onc.2012.165

61. Siddiqui-Jain A, Drygin D, Streiner N, et al. CX-4945, an orally bioavailable selective inhibitor of protein kinase CK2, inhibits prosurvival and angiogenic signaling and exhibits antitumor efficacy. Cancer Res. 2010; 70(24): 10288-10298. doi: 10.1158/0008-5472.CAN-10-1893

62. Pierre F, Chua PC, O’Brien SE, et al. Pre-clinical characterization of CX-4945, a potent and selective small molecule inhibitor of CK2 for the treatment of cancer. Mol Cell Biochem. 2011; 356(1-2): 37-43. doi: 10.1007/s11010-011-0956-5

63. Siddiqui-Jain A, Bliesath J, Macalino D, et al. CK2 inhibitor CX-4945 suppresses DNA repair response triggered by DNAtargeted anticancer drugs and augments efficacy: mechanistic rationale for drug combination therapy. Mol Cancer Ther. 2012; 11(4): 994-1005. doi: 10.1158/1535-7163.MCT-11-0613

64. Song DH, Sussman DJ, Seldin DC. Endogenous protein kinase CK2 participates in Wnt signaling in mammary epithelial cells. J Biol Chem. 2000; 275(31): 23790-23797. doi: 10.1074/ jbc.M909107199

65. Liu LZ, Fang J, Zhou Q, Hu X, Shi X, Jiang BH. Apigenin inhibits expression of vascular endothelial growth factor and angiogenesis in human lung cancer cells: implication of chemoprevention of lung cancer. Mol Pharmacol. 2005; 68(3): 635-643. doi: 10.1124/mol.105.011254

66. Shukla S, Mishra A, Fu P, MacLennan GT, Resnick MI, Gupta S. Up-regulation of insulin-like growth factor binding protein-3 by apigenin leads to growth inhibition and apoptosis of 22Rv1 xenograft in athymic nude mice. FASEB J. 2005; 19(14): 2042-2044. doi: 10.1096/fj.053740fje

67. Fang J, Zhou Q, Liu LZ, et al. Apigenin inhibits tumor angiogenesis through decreasing HIF-1alpha and VEGF expression. Carcinogenesis. 2007; 28(4): 858-864. doi: 10.1093/carcin/bgl205

68. Hu XW, Meng D, Fang J. Apigenin inhibited migration and invasion of human ovarian cancer A2780 cells through focal adhesion kinase. Carcinogenesis. 2008; 29(12): 2369-2376. doi: 10.1093/carcin/bgn244

69. Lamy S, Bedard V, Labbe D, et al. The dietary flavones apigenin and luteolin impair smooth muscle cell migration and VEGF expression through inhibition of PDGFR-beta phosphorylation. Cancer Prev Res (Phila). 2008; 1(6): 452-459. doi: 10.1158/1940-6207.CAPR-08-0072

70. King JC, Lu QY, Li G, et al. Evidence for activation of mutated p53 by apigenin in human pancreatic cancer. Biochim Biophys Acta. 2012; 1823(2): 593-604. doi: 10.1016/j. bbamcr.2011.12.008

71. Torkin R, Lavoie JF, Kaplan DR, Yeger H. Induction of caspase-dependent, p53-mediated apoptosis by apigenin in human neuroblastoma. Mol Cancer Ther. 2005; 4(1): 1-11.

72. Shukla S, Gupta S. Molecular targets for apigenin-induced cell cycle arrest and apoptosis in prostate cancer cell xenograft. Mol Cancer Ther. 2006; 5(4): 843-852. doi: 10.1158/1535-7163. MCT-05-0370

73. Chen D, Landis-Piwowar KR, Chen MS, Dou QP. Inhibition of proteasome activity by the dietary flavonoid apigenin is associated with growth inhibition in cultured breast cancer cells and xenografts. Breast Cancer Res. 2007; 9(6): R80. doi: 10.1186/ bcr1797

74. Kaur P, Shukla S, Gupta S. Plant flavonoid apigenin inactivates Akt to trigger apoptosis in human prostate cancer: an in vitro and in vivo study. Carcinogenesis. 2008; 29(11): 2210-2217. doi: 10.1093/carcin/bgn201

75. Shukla S, Gupta S. Apigenin suppresses insulin-like growth factor I receptor signaling in human prostate cancer: an in vitro and in vivo study. Mol Carcinog. 2009; 48(3): 243-252. doi: 10.1002/mc.20475

76. Cai J, Zhao XL, Liu AW, Nian H, Zhang SH. Apigenin inhibits hepatoma cell growth through alteration of gene expression patterns. Phytomedicine. 2011; 18(5): 366-373. doi: 10.1016/j.phymed.2010.08.006

77. Wang QR, Yao XQ, Wen G, et al. Apigenin suppresses the growth of colorectal cancer xenografts via phosphorylation and up-regulated FADD expression. Oncol Lett. 2011; 2(1): 43-47.doi: 10.3892/ol.2010.215

78. Pandey M, Kaur P, Shukla S, Abbas A, Fu P, Gupta S. Plant flavone apigenin inhibits HDAC and remodels chromatin to induce growth arrest and apoptosis in human prostate cancer cells: in vitro and in vivo study. Mol Carcinog. 2012; 51(12): 952-962. doi: 10.1002/mc.20866

79. Budhraja A, Gao N, Zhang Z, et al. Apigenin induces apoptosis in human leukemia cells and exhibits anti-leukemic activity in vivo. Mol Cancer Ther. 2012; 11(1): 132-142. doi: 10.1158/1535-7163.MCT-11-0343

80. Mafuvadze B, Liang Y, Besch-Williford C, Zhang X, Hyder SM. Apigenin induces apoptosis and blocks growth of medroxyprogesterone acetate-dependent BT-474 xenograft tumors. Horm Cancer. 2012; 3(4): 160-171. doi: 10.1007/s12672-012-0114-x

81. Gao AM, Ke ZP, Wang JN, Yang JY, Chen SY, Chen H. Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/ Nrf2 pathway. Carcinogenesis. 2013; 34(8): 1806-1814. doi: 10.1093/carcin/bgt108

82. Hussain AR, Khan AS, Ahmed SO, et al. Apigenin induces apoptosis via downregulation of S-phase kinase-associated protein 2-mediated induction of p27Kip1 in primary effusion lymphoma cells. Cell Prolif. 2010; 43(2): 170-183. doi: 10.1111/ j.1365-2184.2009.00662.x

83. He J, Xu Q, Wang M, et al. Oral Administration of Apigenin Inhibits Metastasis through AKT/P70S6K1/MMP-9 Pathway in Orthotopic Ovarian Tumor Model. Int J Mol Sci. 2012; 13(6): 7271-7282. doi: 10.3390/ijms13067271

84. Tong X, Pelling JC. Targeting the PI3K/Akt/mTOR axis by apigenin for cancer prevention. Anticancer Agents Med Chem. 2013; 13(7): 971-978. doi: 10.2174/18715206113139990119

85. Gao AM, Ke ZP, Wang JN, Yang JY, Chen SY, Chen H. Apigenin sensitizes doxorubicin-resistant hepatocellular carcinoma BEL-7402/ADM cells to doxorubicin via inhibiting PI3K/Akt/ Nrf2 pathway. Carcinogenesis. 2013; 34(8): 1806-1814. doi: 10.1093/carcin/bgt108

86. Chunhua L, Donglan L, Xiuqiong F, et al. Apigenin upregulates transgelin and inhibits invasion and migration of colorectal cancer through decreased phosphorylation of AKT. J Nutr Biochem. 2013; 24(10): 1766-1775. doi: 10.1016/j. jnutbio.2013.03.006

87. Osada M, Imaoka S, Funae Y. Apigenin suppresses the expression of VEGF, an important factor for angiogenesis, in endothelial cells via degradation of HIF-1alpha protein. FEBS Lett. 2004; 575(1-3): 59-63.doi: 10.1016/j.febslet.2004.08.036

88. Mottet D, Ruys SP, Demazy C, Raes M, Michiels C. Role for casein kinase 2 in the regulation of HIF-1 activity. Int J Cancer. 2005; 117(5): 764-774. doi: 10.1002/ijc.21268

89. Fang J, Zhou Q, Liu LZ, et al. Apigenin inhibits tumor angiogenesis through decreasing HIF-1alpha and VEGF expression. Carcinogenesis. 2007; 28(4): 858-864. doi: 10.1093/carcin/bgl205

90. Mirzoeva S, Kim ND, Chiu K, Franzen CA, Bergan RC, Pelling JC. Inhibition of HIF-1 alpha and VEGF expression by the chemopreventive bioflavonoid apigenin is accompanied by Akt inhibition in human prostate carcinoma PC3-M cells. Mol Carcinog. 2008; 47(9): 686-700. doi: 10.1002/mc.20421

91. Melstrom LG, Salabat MR, Ding XZ, et al. Apigenin downregulates the hypoxia response genes: HIF-1alpha, GLUT-1, and VEGF in human pancreatic cancer cells. J Surg Res. 2011; 167(2): 173-181. doi: 10.1016/j.jss.2010.10.041

92. Gupta S, Afaq F, Mukhtar H. Involvement of nuclear factorkappa B, Bax and Bcl-2 in induction of cell cycle arrest and apoptosis by apigenin in human prostate carcinoma cells. Oncogene. 2002; 21(23): 3727-3738. doi: 10.1038/sj/onc/1205474

93. Lee SH, Ryu JK, Lee KY, et al. Enhanced anti-tumor effect of combination therapy with gemcitabine and apigenin in pancreatic cancer. Cancer Lett. 2008; 259(1): 39-49. doi: 10.1016/j.canlet.2007.09.015

94. Seo HS, Choi HS, Kim SR, et al. Apigenin induces apoptosis via extrinsic pathway, inducing p53 and inhibiting STAT3 and NFkappaB signaling in HER2-overexpressing breast cancer cells. Mol Cell Biochem. 2012; 366(1-2): 319-334. doi: 10.1007/ s11010-012-1310-2

95. Johnson JL, Gonzalez de ME. Interactions between dietary flavonoids apigenin or luteolin and chemotherapeutic drugs to potentiate anti-proliferative effect on human pancreatic cancer cells, in vitro. Food Chem Toxicol. 2013; 60: 83-91. doi: 10.1016/j.fct.2013.07.036

96. Lamy S, Akla N, Ouanouki A, Lord-Dufour S, Beliveau R. Diet-derived polyphenols inhibit angiogenesis by modulating the interleukin-6/STAT3 pathway. Exp Cell Res. 2012; 318(13): 1586-1596. doi: 10.1016/j.yexcr.2012.04.004

97. Song DH, Sussman DJ, Seldin DC. Endogenous protein kinase CK2 participates in Wnt signaling in mammary epithelial cells. J Biol Chem. 2000; 275(31): 23790-23797. doi: 10.1074/ jbc.M909107199

98. Shukla S, MacLennan GT, Flask CA, et al. Blockade of betacatenin signaling by plant flavonoid apigenin suppresses prostate carcinogenesis in TRAMP mice. Cancer Res. 2007; 67(14): 6925-6935. doi: 10.1158/0008-5472.CAN-07-0717

99. Slusarz A, Shenouda NS, Sakla MS, et al. Common botanical compounds inhibit the hedgehog signaling pathway in prostate cancer. Cancer Res. 2010; 70(8): 3382-3390. doi: 10.1158/0008- 5472.CAN-09-3012

100. Tsai CH, Lin FM, Yang YC, et al. Herbal extract of Wedelia chinensis attenuates androgen receptor activity and orthotopic growth of prostate cancer in nude mice. Clin Cancer Res. 2009; 15(17): 5435-5444. doi: 10.1158/1078-0432.CCR-09-0298

101. Lin FM, Chen LR, Lin EH, et al. Compounds from Wedelia chinensis synergistically suppress androgen activity and growth in prostate cancer cells. Carcinogenesis. 2007; 28(12): 2521- 2529. doi: 10.1093/carcin/bgm137

102. Cai X, Liu X. Inhibition of Thr-55 phosphorylation restores p53 nuclear localization and sensitizes cancer cells to DNA damage. Proc Natl Acad Sci U S A. 2008; 105(44): 16958-16963. doi: 10.1073/pnas.0804608105

103. Zhong Y, Krisanapun C, Lee SH, et al. Molecular targets of apigenin in colorectal cancer cells: involvement of p21, NAG-1 and p53. Eur J Cancer. 2010; 46(18): 3365-3374. doi: 10.1016/j.ejca.2010.07.007

104. Dixit D, Sharma V, Ghosh S, Mehta VS, Sen E. Inhibition of Casein kinase-2 induces p53-dependent cell cycle arrest and sensitizes glioblastoma cells to tumor necrosis factor (TNFalpha)-induced apoptosis through SIRT1 inhibition. Cell Death Dis. 2012; 3: e271. doi: 10.1038/cddis.2012.10

105. Choi EJ, Kim GH. Apigenin causes G(2)/M arrest associated with the modulation of p21(Cip1and Cdc2 and activates p53- dependent apoptosis pathway in human breast cancer SK-BR-3 cells. J Nutr Biochem. 2009; 20(4): 285-290. doi: 10.1016/j.jnutbio.2008.03.005

106. Shukla S, Gupta S. Apigenin-induced prostate cancer cell death is initiated by reactive oxygen species and p53 activation. Free Radic Biol Med. 2008; 44(10): 1833-1845. doi: 10.1016/j.freeradbiomed.2008.02.007

107. Chiang LC, Ng LT, Lin IC, Kuo PL, Lin CC. Anti-proliferative effect of apigenin and its apoptotic induction in human Hep G2 cells. Cancer Lett. 2006; 237(2): 207-214. doi: 10.1016/j. canlet.2005.06.002

108. Zheng PW, Chiang LC, Lin CC. Apigenin induced apoptosis through p53-dependent pathway in human cervical carcinoma cells. Life Sci. 2005; 76(12): 1367-1379. doi: 10.1016/j. lfs.2004.08.023

109. Takagaki N, Sowa Y, Oki T, Nakanishi R, Yogosawa S, Sakai T. Apigenin induces cell cycle arrest and p21/WAF1 expression in a p53-independent pathway. Int J Oncol. 2005; 26(1): 185-189. doi: 10.3892/ijo.26.1.185

110. Lolli G, Cozza G, Mazzorana M, et al. Inhibition of protein kinase CK2 by flavonoids and tyrphostins. A structural insight. Biochemistry. 2012; 51(31): 6097-6107. doi: 10.1021/bi300531c

111. Attoub S, Hassan AH, Vanhoecke B, et al. Inhibition of cell survival, invasion, tumor growth and histone deacetylase activity by the dietary flavonoid luteolin in human epithelioid cancer cells. Eur J Pharmacol. 2011; 651(1-3): 18-25. doi: 10.1016/j. ejphar.2010.10.063

112. Yang SF, Yang WE, Chang HR, Chu SC, Hsieh YS. Luteolin induces apoptosis in oral squamous cancer cells. J Dent Res. 2008; 87(4): 401-406. doi: 10.1177/154405910808700413

113. Chiu FL, Lin JK. Downregulation of androgen receptor expression by luteolin causes inhibition of cell proliferation and induction of apoptosis in human prostate cancer cells and xenografts. Prostate. 2008; 68(1): 61-71. doi: 10.1002/pros.20690

114. Bagli E, Stefaniotou M, Morbidelli L, et al. Luteolin inhibits vascular endothelial growth factor-induced angiogenesis; inhibition of endothelial cell survival and proliferation by targeting phosphatidylinositol 3’-kinase activity. Cancer Res. 2004; 64(21): 7936-7946. doi: 10.1158/0008-5472.CAN-03-3104

115. Selvendiran K, Koga H, Ueno T, et al. Luteolin promotes degradation in signal transducer and activator of transcription 3 in human hepatoma cells: an implication for the antitumor potential of flavonoids. Cancer Res. 2006; 66(9): 4826-4834. doi: 10.1158/0008-5472.CAN-05-4062

116. Shi R, Huang Q, Zhu X, et al. Luteolin sensitizes the anticancer effect of cisplatin via c-Jun NH2-terminal kinase-mediated p53 phosphorylation and stabilization. Mol Cancer Ther. 2007; 6(4): 1338-1347. doi: 10.1158/1535-7163.MCT-06-0638

117. Zhou Q, Yan B, Hu X, Li XB, Zhang J, Fang J. Luteolin inhibits invasion of prostate cancer PC3 cells through E-cadherin. Mol Cancer Ther. 2009; 8(6): 1684-1691. doi: 10.1158/1535- 7163.MCT-09-0191

118. Hwang JT, Park OJ, Lee YK, et al. Anti-tumor effect of luteolin is accompanied by AMP-activated protein kinase and nuclear factor-kappaB modulation in HepG2 hepatocarcinoma cells. Int J Mol Med. 2011; 28(1): 25-31. doi: 10.3892/ijmm.2011.667

119. Tsai CH, Lin FM, Yang YC, et al. Herbal extract of Wedelia chinensis attenuates androgen receptor activity and orthotopic growth of prostate cancer in nude mice. Clin Cancer Res. 2009; 15(17): 5435-5444. doi: 10.1158/1078-0432.CCR-09-0298

120. Yan J, Wang Q, Zheng X, et al. Luteolin enhances TNFrelated apoptosis-inducing ligand’s anticancer activity in a lung cancer xenograft mouse model. Biochem Biophys Res Commun. 2012; 417(2): 842-846. doi: 10.1016/j.bbrc.2011.12.055

121. Lee EJ, Oh SY, Sung MK. Luteolin exerts anti-tumor activity through the suppression of epidermal growth factor receptor-mediated pathway in MDA-MB-231 ER-negative breast cancer cells. Food Chem Toxicol. 2012; 50(11): 4136-4143. doi: 10.1016/j.fct.2012.08.025

122. Pratheeshkumar P, Son YO, Budhraja A, et al. Luteolin inhibits human prostate tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis. PLoS ONE. 2012; 7(12): e52279. doi: 10.1371/journal.pone.0052279

123. Huang WW, Chiu YJ, Fan MJ, et al. Kaempferol induced apoptosis via endoplasmic reticulum stress and mitochondriadependent pathway in human osteosarcoma U-2 OS cells. Mol Nutr Food Res. 2010; 54(11): 1585-1595. doi: 10.1002/ mnfr.201000005

124. Khan N, Asim M, Afaq F, Abu ZM, Mukhtar H. A novel dietary flavonoid fisetin inhibits androgen receptor signaling and tumor growth in athymic nude mice. Cancer Res. 2008; 68(20): 8555-8563. doi: 10.1158/0008-5472.CAN-08-0240

125. Tripathi R, Samadder T, Gupta S, Surolia A, Shaha C. Anticancer activity of a combination of cisplatin and fisetin in embryonal carcinoma cells and xenograft tumors. Mol Cancer Ther. 2011; 10(2): 255-268. doi: 10.1158/1535-7163.MCT-10-0606

126. Ying TH, Yang SF, Tsai SJ, et al. Fisetin induces apoptosis in human cervical cancer HeLa cells through ERK1/2-mediated activation of caspase-8-/caspase-3-dependent pathway. Arch Toxicol. 2012; 86(2): 263-273. doi: 10.1007/s00204-011-0754-6

127. Zhou W, Kallifatidis G, Baumann B, et al. Dietary polyphenol quercetin targets pancreatic cancer stem cells. Int J Oncol. 2010; 37(3): 551-561. doi: 10.3892/ijo_00000704

128. Zhong X, Wu K, He S, Ma S, Kong L. Effects of quercetin on the proliferation and apoptosis in transplantation tumor of breast cancer in nude mice. Sichuan Da Xue Xue Bao Yi Xue Ban. 2003; 34(3): 439-442.

129. Ma ZS, Huynh TH, Ng CP, Do PT, Nguyen TH, Huynh H. Reduction of CWR22 prostate tumor xenograft growth by combined tamoxifen-quercetin treatment is associated with inhibition of angiogenesis and cellular proliferation. Int J Oncol. 2004; 24(5): 1297-1304. doi: 10.3892/ijo.24.5.1297

130. Dechsupa S, Kothan S, Vergote J, et al. Quercetin, Siamois 1 and Siamois 2 induce apoptosis in human breast cancer MDAmB-435 cells xenograft in vivo. Cancer Biol Ther. 2007; 6(1): 56-61. doi: 10.4161/cbt.6.1.3548

131. Sun ZJ, Chen G, Hu X, et al. Activation of PI3K/Akt/IKKalpha/NF-kappaB signaling pathway is required for the apoptosis-evasion in human salivary adenoid cystic carcinoma: its inhibition by quercetin. Apoptosis. 2010; 15(7): 850-863. doi: 10.1007/s10495-010-0497-5

132. Cheng S, Gao N, Zhang Z, et al. Quercetin induces tumorselective apoptosis through downregulation of Mcl-1 and activation of Bax. Clin Cancer Res. 2010; 16(23): 5679-5691. doi: 10.1158/1078-0432.CCR-10-1565

133. Long Q, Xiel Y, Huang Y, et al. Induction of apoptosis and inhibition of angiogenesis by PEGylated liposomal quercetin in both cisplatin-sensitive and cisplatin-resistant ovarian cancers. J Biomed Nanotechnol. 2013; 9(6): 965-975. doi: http://dx.doi.org/10.1166/jbn.2013.1596

134. Huang CY, Chan CY, Chou IT, Lien CH, Hung HC, Lee MF. Quercetin induces growth arrest through activation of FOXO1 transcription factor in EGFR-overexpressing oral cancer cells. J Nutr Biochem. 2013; 24(9): 1596-1603. doi: 10.1016/j. jnutbio.2013.01.010

135. Chan ST, Yang NC, Huang CS, Liao JW, Yeh SL. Quercetin enhances the antitumor activity of trichostatin A through upregulation of p53 protein expression in vitro and in vivo. PLoS ONE. 2013; 8(1): e54255. doi: 10.1371/journal.pone.0054255

136. Wang G, Zhang J, Liu L, Sharma S, Dong Q. Quercetin potentiates doxorubicin mediated antitumor effects against liver cancer through p53/Bcl-xl. PLoS ONE. 2012; 7(12): e51764. doi: 10.1371/journal.pone.0051764

137. Pratheeshkumar P, Budhraja A, Son YO, et al. Quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting V EGFR- 2 regulated AKT/mTOR/P70S6K signaling pathways. PLoS ONE. 2012; 7(10): e47516. doi: 10.1371/journal.pone.0047516

138. Gao X, Wang B, Wei X, et al. Anticancer effect and mechanism of polymer micelle-encapsulated quercetin on ovarian cancer. Nanoscale. 2012 21; 4(22): 7021-7030. doi: 10.1039/ c2nr32181e

139. Angst E, Park JL, Moro A, et al. The flavonoid quercetin inhibits pancreatic cancer growth in vitro and in vivo. Pancreas. 2013; 42(2): 223-229. doi: 10.1097/MPA.0b013e318264ccae

140. Kim HS, Wannatung T, Lee S, et al. Quercetin enhances hypoxia-mediated apoptosis via direct inhibition of AMPK activity in HCT116 colon cancer. Apoptosis. 2012; 17(9): 938-949. doi: 10.1007/s104950120719-0

141. Tan BJ, Liu Y, Chang KL, Lim BK, Chiu GN. Perorally active nanomicellar formulation of quercetin in the treatment of lung cancer. Int J Nanomedicine. 2012; 7: 651-661. doi: 10.2147/ IJN.S26538

142. Zheng SY, Li Y, Jiang D, Zhao J, Ge JF. Anticancer effect and apoptosis induction by quercetin in the human lung cancer cell line A-549. Mol Med Rep. 2012; 5(3): 822-826. doi: 10.3892/mmr.2011.726

143. Lin C, Yu Y, Zhao HG, Yang A, Yan H, Cui Y. Combination of quercetin with radiotherapy enhances tumor radiosensitivity in vitro and in vivo. Radiother Oncol. 2012; 104(3): 395-400. doi: 10.1016/j.radonc.2011.10.023

144. Wang K, Liu R, Li J, et al. Quercetin induces protective autophagy in gastric cancer cells: involvement of Akt-mTOR- and hypoxia-induced factor 1alpha-mediated signaling. Autophagy. 2011; 7(9): 966-978.doi: 10.4161/auto.7.9.15863

145. Wong MY, Chiu GN. Liposome formulation of co-encapsulated vincristine and quercetin enhanced antitumor activity in a trastuzumab-insensitive breast tumor xenograft model. Nanomedicine. 2011; 7(6): 834-840. doi: 10.1016/j.nano.2011.02.001

146. Sun F, Zheng XY, Ye J, Wu TT, Wang J, Chen W. Potential anticancer activity of myricetin in human T24 bladder cancer cells both in vitro and in vivo. Nutr Cancer. 2012; 64(4): 599- 606. doi: 10.1080/01635581.2012.665564

147. Valentova K, Vrba J, Bancirova M, Ulrichova J, Kren V. Isoquercitrin: pharmacology, toxicology, and metabolism. Food Chem Toxicol. 2014; 68: 267-282. doi: 10.1016/j. fct.2014.03.018

148. Murota K, Matsuda N, Kashino Y, et al. alpha-Oligoglucosylation of a sugar moiety enhances the bioavailability of quercetin glucosides in humans. Arch Biochem Biophys. 2010; 501(1): 91-97. doi: 10.1016/j.abb.2010.06.036

149. Makino T, Shimizu R, Kanemaru M, Suzuki Y, Moriwaki M, Mizukami H. Enzymatically modified isoquercitrin, alphaoligoglucosyl quercetin 3-O-glucoside, is absorbed more easily than other quercetin glycosides or aglycone after oral administration in rats. Biol Pharm Bull. 2009; 32(12): 2034-2040.doi: 10.1248/bpb.32.2034

150. Kawai M, Hirano T, Arimitsu J, et al. Effect of enzymatically modified isoquercitrin, a flavonoid, on symptoms of Japanese cedar pollinosis: a randomized double-blind placebo-controlled trial. Int Arch Allergy Immunol. 2009; 149(4): 359-368. doi: 10.1159/000205582

151. Motoyama K, Koyama H, Moriwaki M, et al. Atheroprotective and plaque-stabilizing effects of enzymatically modified isoquercitrin in atherogenic apoE-deficient mice. Nutrition. 2009; 25(4): 421-427. doi: 10.1016/j.nut.2008.08.013

152. Shia CS, Tsai SY, Kuo SC, Hou YC, Chao PD. Metabolism and pharmacokinetics of 3,3’,4’,7-tetrahydroxyflavone (fisetin), 5-hydroxyflavone, and 7-hydroxyflavone and antihemolysis effects of fisetin and its serum metabolites. J Agric Food Chem. 2009; 57(1): 83-89. doi: 10.1021/jf802378q

153. FISHMAN WH, ANLYAN AJ. The presence of high beta-glucuronidase activity in cancer tissue. J Biol Chem. 1947; 169(2): 449.

154. Bosslet K, Straub R, Blumrich M, et al. Elucidation of the mechanism enabling tumor selective prodrug monotherapy. Cancer Res. 1998; 58(6): 1195-1201

155. Paigen K. Mammalian beta-glucuronidase: genetics, molecular biology, and cell biology. Prog Nucleic Acid Res Mol Biol. 1989; 37: 155-205.doi: 10.1016/s0079-6603(08)60698-4

156. Estrella V, Chen T, Lloyd M, et al. Acidity generated by the tumor microenvironment drives local invasion. Cancer Res. 2013; 73(5): 1524-1535. doi: 10.1158/0008-5472.CAN-12- 2796

157. Murdter TE, Friedel G, Backman JT, et al. Dose optimization of a doxorubicin prodrug (HMR 1826in isolated perfused human lungs: low tumor pH promotes prodrug activation by beta-glucuronidase. J Pharmacol Exp Ther. 2002; 301(1): 223- 228. doi: 10.1124/jpet.301.1.223

158. Sperker B, Werner U, Murdter TE, et al. Expression and function of beta-glucuronidase in pancreatic cancer: potential role in drug targeting. Naunyn Schmiedebergs Arch Pharmacol. 2000; 362(2): 110-115. doi: 10.1007/s002100000260

159. de GM, Boven E, Scheeren HW, Haisma HJ, Pinedo HM. Beta-glucuronidase-mediated drug release. Curr Pharm Des. 2002; 8(15): 1391-1403. doi: 10.2174/1381612023394485

160. Yuan L, Wagatsuma C, Yoshida M, et al. Inhibition of human breast cancer growth by GCP (genistein combined polysaccharidein xenogeneic athymic mice: involvement of genistein biotransformation by beta-glucuronidase from tumor tissues. Mutat Res. 2003; 523-524: 55-62. doi: 10.1016/s0027-5107- (02)00321-4

161. Chen X, Wu B, Wang PG. Glucuronides in anti-cancer therapy. Curr Med Chem Anticancer Agents. 2003; 3(2): 139-150. doi: 10.2174/1568011033353470

162. Oi N, Hashimoto T, Kanazawa K. Metabolic conversion of dietary quercetin from its conjugate to active aglycone following the induction of hepatocarcinogenesis in fisher 344 rats. J Agric Food Chem. 2008; 56(2): 577-583. doi: 10.1021/jf072556c

163. Murakami A, Ashida H, Terao J. Multitargeted cancer prevention by quercetin. Cancer Lett. 2008; 269(2): 315-325. doi: 10.1016/j.canlet.2008.03.046

164. Legigan T, Clarhaut J, Renoux B, et al. Synthesis and antitumor efficacy of a beta-glucuronidase-responsive albuminbinding prodrug of doxorubicin. J Med Chem. 2012; 55(9): 4516-4520.doi: 10.1021/jm300348r

165. Chen KC, Schmuck K, Tietze LF, Roffler SR. Selective cancer therapy by extracellular activation of a highly potent glycosidic duocarmycin analogue. Mol Pharm. 2013; 10(5): 1773- 1782. doi: 10.1021/mp300581u

166. Myzak MC, Tong P, Dashwood WM, Dashwood RH, Ho E. Sulforaphane retards the growth of human PC-3 xenografts and inhibits HDAC activity in human subjects. Exp Biol Med (wood). 2007; 232(2): 227-234.

167. Bali P, Pranpat M, Bradner J, et al. Inhibition of histone deacetylase 6 acetylates and disrupts the chaperone function of heat shock protein 90: a novel basis for antileukemia activity of histone deacetylase inhibitors. J Biol Chem. 2005; 280(29): 26729-26734. doi: 10.1074/jbc.C500186200

168. Aoyagi S, Archer TK. Modulating molecular chaperone Hsp90 functions through reversible acetylation. Trends Cell Biol. 2005; 15(11): 565-567. doi: 10.1016/j.tcb.2005.09.003

169. Rao R, Fiskus W, Yang Y, et al. HDAC6 inhibition enhances 17-AAG--mediated abrogation of hsp90 chaperone function in human leukemia cells. Blood. 2008; 112(5): 1886-1893. doi: 10.1182/blood2008-03-143644

170. Ai J, Wang Y, Dar JA, et al. HDAC6 regulates androgen receptor hypersensitivity and nuclear localization via modulating Hsp90 acetylation in castration-resistant prostate cancer. Mol Endocrinol. 2009; 23(12): 1963-1972. doi: 10.1210/me.2009- 0188

171. Gibbs A, Schwartzman J, Deng V, Alumkal J. Sulforaphane destabilizes the androgen receptor in prostate cancer cells by inactivating histone deacetylase 6. Proc Natl Acad Sci U S A. 2009; 106(39): 16663-16668. doi: 10.1073/pnas.0908908106

172. Aldana-Masangkay GI, Sakamoto KM. The role of HDAC6 in cancer. J Biomed Biotechnol. 2011; 2011: 875824. doi: 10.1155/2011/875824

173. Kaliszczak M, Trousil S, Aberg O, Perumal M, Nguyen QD, Aboagye EO. A novel small molecule hydroxamate preferentially inhibits HDAC6 activity and tumour growth. Br J Cancer. 2013; 108(2): 342-350. doi: 10.1038/bjc.2012.576

174. Wang P, Henning SM, Heber D, Vadgama JV. Sensitization to docetaxel in prostate cancer cells by green tea and quercetin. J Nutr Biochem. 2015. doi: 10.1016/j.jnutbio.2014.11.017

175. Chen FY, Cao LF, Wan HX, et al. Quercetin enhances adriamycin cytotoxicity through induction of apoptosis and regulation of mitogen-activated protein kinase/extracellular signal-regulated kinase/c-Jun N-terminal kinase signaling in multidrug-resistant leukemia K562 cells. Mol Med Rep. 2015; 11(1): 341-348. doi: 10.3892/mmr.2014.2734

176. Maciejczyk A, Surowiak P. Quercetin inhibits proliferation and increases sensitivity of ovarian cancer cells to cisplatin and paclitaxel. Ginekol Pol. 2013; 84(7): 590-595.doi: 10.17772/gp/1609

177. Han Y, Yu H, Wang J, Ren Y, Su X, Shi Y. Quercetin alleviates myocyte toxic and sensitizes anti-leukemic effect of adriamycin. Hematology. 2014. doi: http://dx.doi.org/10.1179/1607845414y.0000000198

178. Orsolic N, Car N. Quercetin and hyperthermia modulate cisplatin-induced DNA damage in tumor and normal tissues in vivo. Tumour Biol. 2014; 35(7): 6445-6454. doi: 10.1007/ s13277-014-1843-y

179. Papiez MA. The effect of quercetin on oxidative DNA damage and myelosuppression induced by etoposide in bone marrow cells of rats. Acta Biochim Pol. 2014; 61(1): 7-11.

180. Mahoney SE, Davis JM, Murphy EA, McClellan JL, Pena MM. Dietary quercetin reduces chemotherapy-induced fatigue in mice. Integr Cancer Ther. 2014; 13(5): 417-424. doi: 10.1177/1534735414523315

181. Azevedo MI, Pereira AF, Nogueira RB, et al. The antioxidant effects of the flavonoids rutin and quercetin inhibit oxaliplatin-induced chronic painful peripheral neuropathy. Mol Pain. 2013; 9: 53. doi: 10.1186/1744-8069-9-53

182. Lou DY, Dominguez I, Toselli P, Landesman-Bollag E, O’Brien C, Seldin DC. The alpha catalytic subunit of protein kinase CK2 is required for mouse embryonic development. Mol Cell Biol. 2008; 28(1): 131-139. doi: 10.1128/MCB.01119-07

183. Landesman-Bollag E, Belkina A, Hovey B, Connors E, Cox C, Seldin DC. Developmental and growth defects in mice with combined deficiency of CK2 catalytic genes. Mol Cell Biochem. 2011; 356(1-2): 227-231. doi: 10.1007/s11010-011-0967-2

184. Hasumura M, Yasuhara K, Tamura T, Imai T, Mitsumori K, Hirose M. Evaluation of the toxicity of enzymatically decomposed rutin with 13-weeks dietary administration to Wistar rats. Food Chem Toxicol. 2004; 42(3): 439-444. doi: 10.1016/j. fct.2003.10.006

LATEST ARTICLES

Laparoscopic Management of Adrenal and Extra-Adrenal

Laparoscopic Management of Adrenal and Extra-Adrenal Pheochromocytoma

Shrenik J. Shah*, Sajid Nurbhai, Rusha Surti, Parixit Malaviya and Pratik Chaudhary

doi.10.17140/UAOJ-7-146

Penile Cancer in the Region of Thies

Penile Cancer in the Region of Thies: Epidemiological, Diagnostic and Therapeutic Aspects

Saint C. N. Kouka*, Tonleu L. Bentefouet, Ngor M. Thiam, Modou Faye, Mbayang Diop, Mouhamed Cisse, Mohamed Jalloh, Aissatou A. Diame, Yoro Diallo and Sylla Cheikhna

doi.10.17140/UAOJ-7-145

Coronavirus Disease-2019 Infection-Associated Glomerular Diseases

Phuong-Chi T. Pham*, Golriz Jafari, Anita Kamarzarian, Vinod K. Valluri, Kulwant Bath, Chau Sally, Nguyen Tuan, Mahalli Joseph, Phuong-Mai T. Pham, Phuong-Anh T. Pham, Son V. Pham and Phuong-Thu T. Pham

doi.10.17140/NPOJ-8-129

West Virginia University Medicine, Wheeling Hospital’s Sepsis Study

Ramya Ramesh*, Jazmin Jatana, Chan Hong, Sathyanarayana Machani, Milind Awale, Stanley Guertal, Catherine Macalister, Heather L. Merkel, Melissa Burkett and Silvia Myndresku

doi. 10.17140/EMOJ-10-173

Yet Another Public Health Threat: A Commentary and Examination of the Extensive Use of Bromazolam

Nelson J. Tiburcio* and Scarlett L. Baker

doi.10.17140/PHOJ-9-167

LATEST ARTICLES