Inhibition of cancer antioxidant defense by natural compounds

All classic, non-surgical anticancer approaches like chemotherapy, radiotherapy or photodynamic therapy kill cancer cells by inducing severe oxidative stress. Even tough chemo- and radiotherapy are still a gold standard in cancer treatment, the identification of non-toxic compounds that enhance their selectivity, would allow for lowering their doses, reduce side effects and risk of second cancers. Many natural products have the ability to sensitize cancer cells to oxidative stress induced by chemo- and radiotherapy by limiting antioxidant capacity of cancer cells. Blocking antioxidant defense in tumors decreases their ability to balance oxidative insult and results in cell death. Though one should bear in mind that the same natural compound often exerts both anti-oxidant and pro-oxidant properties, depending on concentration used, cell type, exposure time and environmental conditions. Here we present a comprehensive overview of natural products that inhibit major antioxidant defense mechanisms in cancer cells and discuss their potential in clinical application.


INTRODUCTION
Over 60% of currently used antitumor drugs come fromatural sources such as plants, fungi and microorganisms. The large scale screening programs foratural products with anticancer activities,.g.hose launched in 1950s bytalianesearch company or in 1960s byhe National Cancernstitute (NCI), allowed for identification of bacteria-produced doxorubicin andaxol (paclitaxel), derived fromhe bark ofhe yewree. Both ofhese compounds are widely used in chemotherapyegimens in different cancerypes. Thoughheir mechanism of action is different as doxorubicin intercalates into DNA and abrogateseplication [1] andaxol inhibits microtubules depolymerization during mitosis [2],hey both induce strong oxidative stress,hough by different means [3][4][5]. Total cellular antioxidant capacity is a known determinant of cancer susceptibilityohese drugs [6][7][8]. Oxidative stress induced by chemotherapeutics is crucial forheirfficacy, but, onhe other hand, contributesohe cumulative and irreversible cardiotoxicity observed clinically [9,10]. These sideffects highlighthe lack of selectivity of chemotherapy [11]. Therefore,on-toxicatural substanceshat potentiate action of chemotherapeutics and allow for loweringheir concentration are of a particular interestohe anticancer drug field.
Higher steady-state levels of ROS in cancer cellselativeoormal cells have been known for around 35 years [17].ncreased ROS are crucial inhe initiation Review www.impactjournals.com/oncotarget of carcinogenesis when acquiringew mutations and clonalxpansion of initiated cells areeededostablish aumor. Thisendershem bothhe cause andheesult of cellularransformation: ROS-induced oxidative damage favors production of moreadicals andstablishes a feed-in loop, increasing mutationsate, activating oncogenes,nhancing metaboliceprogramming and progression ofumors. Thenhanced ROS generation is induced by oncogenic signaling with main drivers: V-Ras, K-Ras, mtp53 and c-Myc [18,19] and involves both mitochondrial and cytoplasmic ROS. K-Rasinduced cellularransformation was shownoequire NOX1 activationhrough p38/PDPK1/PKCδ/p47phox cascade [20], whilexpression of Myr-Akt, H-RasG12V and K-RasG12D in murinembryonic fibroblasts (MEFs) conferred increased mitochondrial ROS-dependent soft agar colony formation [21]. Mutations inumor suppressors genes are often associated withhe induction of strong oxidative stress and promotehe survival of cells with high ROS levels. Mutant BRCA1 and p53 were showno attenuate antioxidant signaling driven byheuclear factor (erythroid-derived 2)-like 2 (Nrf2), contributingo cancer initiation [22,23] One ofhe consequences ofhexcessive damage caused by ROS are changes in mitochondrial membrane permeabilityhatesult in cytochrome Celease and apoptotic death [24][25][26][27][28][29].n defense, cancer cells boostheir antiapoptotic mechanisms likeuclear factor kappa-lightchain-enhancer of activated B cells (NFĸB) pathwayoscape cell death [30,31]. Decreased mitochondrial activityriggershe glycolytic switch and upregulates glycolytic pathway in ordero produce morenergy and biomass (ribose, amino acids, fatty acids) forapidly proliferating cancer cells [32]. Moreover,xposureo oxidative stress induces mutations in mitochondrial DNA as well as in VEGF (Vascular Endothelial Growth Factor) and HIF-1α (Hypoxianducible Factor-1α) genes, promoting angiogenesis and furthernhancing metaboliceprogramming of cells [33]. Oxidative stress also changesheumor microenvironmento support growth Coupled to molecular oxygen they give rise to the primary free radical and the precursor of remaining species -superoxide (·O 2 -). In the reaction with a short-lived nitric oxide (·NO), superoxide forms a highly reactive peroxynitrate (ONOO -) able to modify structure and function of proteins. Alternatively, superoxide dismutase (SOD) converts superoxide to hydrogen peroxide (H 2 O 2 ), which can be further transformed in several ways. In the presence of transition metal ions like Fe 2+ (Fenton's reaction) or in reaction with superoxide, H 2 O 2 forms highly reactive hydroxyl radical (·OH) which damages lipids, proteins and DNA. Peroxysomal enzyme catalase (CAT) neutralizes H 2 O 2 to water and oxygen. H 2 O 2 might be also utilized in the reaction of oxidation of monomeric glutathione (GSH) to the glutathione disulfide (GSSG) or reduced thioredoxin (Trx red ) to the oxidized thioredoxin (Trx ox ) catalyzed by glutathione peroxidase (GPX) or peroxidases involved in the thioredoxin turnover (PRX). Reduced glutathione pool is restored by glutathione reductase (GR) which reduces oxidized glutathione with the use of NADPH. Similarly, thioredoxin reductase (TrxR) balances the amount of reduced Trx by transferring electrons from NADPH to oxidized catalytic sites. Thanks to the thiol groups in the Cys residues both glutathione and thioredoxin participate in the reduction of oxidized proteins. Their synthesis as well as the turnover are under tight homeostatic control creating a system responsible for reduction of redox-sensitive proteins upon oxidative stress. and cell spread. Hydrogen peroxide produced byumorissue can initiate destruction ofon-tumor surroundingissueo obtainutrients and promote growth [34]. Thisxplains whyumors are saido be "addictedo ROS signaling".
Mitochondrial ROS arehe major inducers of autophagy, however, upon chronic impairment of mitochondrial function, highxtent ofadicals shifts signaling into self-removal of mitochondriahrough a selective process called mitophagy [43,44]. This fine mechanism allows autophagyoliminatehe source of oxidative stress and protecthe cell from oxidative damage.

TARGETING ROS ADAPTATIONS IN CANCER
Because ofhis sharpeliance on ROS production, cancer cells are more vulnerableo further disturbance ofheired-ox statushanormal cells. This differencestablishes aherapeutic window allowing for anmergence ofhe selective anticancer strategy based on modulation of cancer cellsedox potential. Dueohenhanced antioxidant capacity ofumors, just inducing ROS generation isot sufficient for a successfulradication of cancer. The drug opposing properties in cancer cells, depending on their concentration. At lower concentrations they often boost cells' antioxidant capacity by activating Nrf2-dependent signaling and enhancing expression of ROS scavengers, lowering ROS burden. These properties allow for using natural compounds in chemoprevention and as agents decreasing side effects of standard anticancer regimens. On the other hand, same compounds used at higher concentrations inhibit antioxidant defense and induce oxidative stress. By doing that they enhance the effectiveness of chemo-and radiotherapy and allow for lowering their doses. www.impactjournals.com/oncotarget  [67] Sensitizes tumor xenografts to doxorubicin [67] Induces glutathione depletion [94] and inhibits mitochondrial complex I activity in rats [151] Chaetocin Polyphenol thiodioxopiperazine

Citrus spp.
Inhibits TrxR leading to death of lung carcinomas [114] Inhibits mitochondrial complex I activity in rats [151] Resveratrol Binds GSH and inhibits its metabolism in leukemias [92] Increases IκBα and suppresses NFκB in human gliomas resulting in ROS-induced apoptosis [93] Trigonelline Alkaloid Pyridine and piperidine group coffee Reduces nuclear accumulation of Nrf2 in pancreatic cancer cells and sensitizes them to anticancer drugs and TRAIL via Nrf2 inhibition [76]. Enhances response to chemotherapy in vivo [76] www.impactjournals.com/oncotarget should also inhibithe antioxidant defense system [48]. Many compounds ofatural origin block Nrf2 pathway or directly inhibitndogenous antioxidants leadingohelevated ROS production. Moreover, Nrf2 inhibitionesults in a decrease of drugffluxransporters and a consequent increase inetention of anticancer drugs in cells. Therefore Nrf2 or cellular antioxidant inhibitors synergize with classic chemotherapeutics and decreaseheiroxicity. Surprisingly, amonghemhere are polyphenols likeesveratrol, quercetin, EGCG, apigenin, luteolin or chrysin which were initiallyeportedo have ROS scavenging properties and are generallyecognized as antioxidants. Therefore a considerable caution should bexercised when applyingatural products as adjuvants sinceheirffects strongly depend on concentration, cellype,xposureime andnvironmental conditions [49-55].

THE CELLULAR ANTIOXIDANT DEFENSE
Increased levels of freeadicalsnableumor cellso activate pathways driving proliferation, angiogenesis, metastasis andhrive under hypoxic conditions [79][80][81]. High levels of ROS createheisk of damage linkedo oxidative stress,herefore cancer cellsendo overexpress detoxifying proteinshatlevateheir antioxidant capacity. Hyper-activation of Nrf2 pathway increaseshe amount of cellular ROS scavengers. Lowering stress burden by means ofnhancing detoxifying force further affects certain pathwayshat promote growth and proliferation [82][83][84]. Blocking antioxidant activity in cancer cells decreasesheir abilityo balance oxidative insult and mightesult in cell death [85]. Below are presented key cellular antioxidant systems andatural compounds disturbingheir activity GSH One ofhe major systems involved inesponseo freeadicalselies on aripeptide -glutathione. The sulfhydryl (SH) group ofeduced glutathione accounts for its stronglectron-donating properties (Figure 1). Once oxidized,wo glutathione molecules form a dimer linked by a disulfide bridge (GSSG). GSHeacts with proteinso form S-glutathionylated proteins, protectinghem from further oxidation. Glutathioneot only directly scavenges freeadicals (hydroxyladical, singlet oxygen), but also serves as a cofactor of several detoxifyingnzymeshatequirehiol-reducingquivalents (glutathione peroxidase, glutathioneransferase). GSH is also involved inecycling other antioxidants byeducing vitamins C and E [86]. Most of cellular GSH contentemains inhe cytosol, however it can also be found in organelles, including mitochondria, peroxisomes,ndoplasmiceticulum andheucleus [87]. Givenhe prominentole in keeping cells'edox homeostasis in check, glutathione metabolism is accelerated in manyypes of cancero alleviate oxidative stress and promote proliferation and metastasis [88]. High levels of GSH are associated with apoptosis-resistant phenotypes and its depletion is linkedohearly stages of cell death initiation [89][90][91]. Nuclear and mitochondrial pool of glutathione plays an importantole in protecting DNA from oxidative stress-driven lesions. Cell death induced by an intercalating drug doxorubicin was potentiated upon glutathione depletion [89]. This might serve as aationaleo designreatment and boostherapeuticffect of anticancer agents.

NCT00192842 Phase II
To assess if curcumin can improve the efficacy of the standard chemotherapy gemcitabine in patients with advanced pancreatic cancer. 5 out of 17 patients (29%) discontinued curcumin due to intractable abdominal fullness or pain, and the dose of curcumin was reduced to 4 mg/day because of abdominal complaints in 2 other patients. One of 11 evaluable patients (9%) had partial response, 4 (36%) had stable disease, and 6 (55%) had tumor progression. [ To determine safety and tolerability of SRT501 in subjects with colorectal cancer and hepatic metastases SRT501 was well tolerated. Mean plasma resveratrol levels following a single dose of SRT501 administration were exceeding those for equivalent doses of nonmicronized resveratrol by 3.6fold. Resveratrol was detectable in hepatic tissue. Cleaved caspase-3 was significantly increased [184]. PEITC (dosage not provided)

NCT00691132
Phase II PEITC in preventing lung cancer in people who smoke The recruitment status unknown for its biological activity and provedoxert anti-cancer properties. PEITC strongly induced oxidative damage dueohe depletion of glutathione and inhibition of GPX in H-Rasransformed ovarianpithelial cells [98]. Depletion of cellular glutathione after PEITCreatment was observed in cancer cells of different origin, including glioma, oral cavity cancer, leukemia, prostate and breast [99][100][101][102][103]. Recent data demonstratehat PEITC caused inhibition of GST in glioma GBM 8401cells, leadingo massive ROS induction and causing cell death [104]. PEITC sensitized cancer cellso cisplatin in biliaryracthrough PEITCinduced depletion of overall GSH, which facilitated Mcl-1 glutathionylation, promoted Mcl-1 degradation andesensitized cellso cisplatin [105]. This data indicatehat combined anticancerherapy based on synergisticffect of GSH depletion and strong oxidative stress induction leadso anffective cancer cell killing.

NATURAL PRODUCTS BLOCKING SOD ACTIVITY
Since mitochondria arehe primary source of cellular freeadicals, decreasingheir detoxifying ability by means of blocking SOD2 activity inumors might contributeohe apoptosis activation. Plumbagin provedofficiently induce apoptosis inffect in prostate cancer cell lines, partiallyhrough decreasing SOD2xpression [97]. PEITC was foundo inhibitxpression of SOD in LN229 glioma cell line, weakening cellular antioxidant defense and causing apoptosis [99]. Suppression of SODnzymatic activity by apigenin in combination with ROS-inducing paclitaxel was foundo sensitize HeLa cellso apoptosis and allowedo lower paclitaxel doses [123].

CATALASE (CAT)
Catalase is a peroxisomalnzymehateutralizes hydrogen peroxide by its decompositiono water and oxygen (Figure 1). High levels of hydrogen peroxide facilitate DNA mutagenesis,herefore under physiological conditions catalase protects cells from oxidative damage. H 2 O 2 also serves as mediator of apoptosis and can modifyegulatory protein complexes, such as Nrf2/Keap1 system. Apart from peroxisomal CAT, malignant cells acquire membrane-associated catalaseo survive under oxidative stress [124][125][126]. Blocking catalase activity can significantly increase oxidative burdenhrough hydrogen peroxide accumulation whichriggeredumor cells death.

NATURAL PRODUCTS INHIBITING CATALASE
Wogonin, a flavonoid isolated from Scutellaria baicalensis was showno induce cell death in cervix, ovary and lung cancer cellshrough catalase inhibitionhat increased hydrogen peroxide levels and facilitated TNFinduced apoptotic signaling [127]. Human hepatoma HepG2 cells subjectedo apigenin accumulated H 2 O 2 , which correlated with a decrease of catalase mRNA and catalase activity and ledo cell death [128]. PEITCreatment lowered catalase protein levels and induced ROS in GBM 8401 glioma cells [104]. EGCG inhibited catalase activity both in vitro and in K562 cells [129] and sensitized cellso arsenite (As)reatment. The proposed mechanismxplainedhathe inhibition of catalase activity uponreatment with As/EGCG occurred via JNK (c-Jun N-terminal kinase) signaling pathway. Genotoxic stresshat activated JNK, promoted catalase phosphorylation by c-Abl kinase, marking it for proteasomal degradation. Blocking catalase activity ledo high amount of H 2 O 2 and promoted death ofpithelial cells subjectedo As/EGCG [130].

EXOGENOUS ANTIOXIDANTS
Theole of oxidative stress in initiating and promoting cancer onhe one hand and in causing oxidative damage onhe other justifieswo opposite ROSmanipulating strategies against cancer. First is antioxidant approach functional in cancer prevention andherapy. The most important and widespreadxogenous dietary antioxidants are vitamins A and E,heir analogs carotenoids andocopherols, vitamin C and polyphenols. Though preventing ROS-induced mutations and subsequent cancer initiation with dietary antioxidants is well documented,heir use during anticancerherapyemains controversial. Since cancerherapy highlyelies onhe production of freeadicals, it has been speculatedhat supplying cells in antioxidants might decreasereatmentfficacy. Onhe other hand,he basic idea behind using antioxidants duringherapy isoliminatexcessive oxidative damage ando help alleviate adverseffects. Many patientseceivingherapy areaking antioxidants without consulting with a physician. Selenium and vitamin C are widely used in complementary oncology [131]. Radiotherapyrials in head andeck cancers showedhat vitamin Eeducedheoxicity, however overallecurrence and mortality wereaised [132,133]. Trials onheffect of antioxidants on chemotherapyeported on some benefits of using vitamin E or selenium with cisplatin,axol and oxiplatin, buthe long-termffects wereot assessed [134][135][136][137][138]. Decreasedecurrence of some cancerypes in patientsoteceivingreatment or after chemotherapy has also beeneported [139,140]. The main conclusion fromheserials ishat administration of antioxidantso cancer patients in combination withherapy should beaken with great care. Patient phenotype (smoking, alcohol uptake andutrition),umor localization (different partial pressures of oxygen amongissues) andype ofherapy should be considered in ordero choose a suitable antioxidant supplement [141].mportantly, adverseffects wereoteported with antioxidants derived from food. The Women's Healthy Eating and Living Study (WHELS), where diet composed of high amount of fruit and vegetable,ich in beta-carotene and vitamin C, showedoffect on outcome in patients witharly breast cancer [142].
Theserials were basing on ROS scavenging properties ofatural compounds. The ability of orally administered EGCGoeducehe incidence and severity ofsophagitis wasested in patients with locally advanced stageIIonsmall-cell lung cancereceiving concurrent chemotherapy andhoracicadiotherapy (phase, NCT01481818). No doselimitingoxicity of EGCG waseported. Dramaticegression ofsophagitiso grade 0/1 was observed in 22 of 24 patients andhe pain score was alsoeduced [143]. Currently,he EGCG-mediated protection ofhesophagus from damage induced byadiotherapy in patients with lung cancer is beingested in phaseI (NCT02577393). Alsoopically administered EGCG wason-toxic and provedffective in decreasingadiation dermatitis in patients with breast cancer after mastectomyeceiving adjuvantadiotherapy [144]. Orally administered curcumin significantlyeducedhe severity of skineactions (dermatitis) caused byadiationherapy breast cancer patients as shown in phaseI/IIIrial (NCT01246973) [145] and prevented colon cancer byeducinghe aberrant crypt foci (ACF)umber in smokers at dose 4 g/day [146]. Unfortunately, just a fewrials so far addressed a question whetheratural compounds could improvehefficacy ofhe standard chemotherapy oradiationherapy. One such arial (phaseI)ested curcumin abilityo potentiateheffect of gemcitabine in patients with advanced pancreatic cancer (NCT00192842).n one out ofwenty one patientsvaluable foresponse curcumin caused brief but markedumoregression (73%) and one patientemained stable for > 18 months. The problem wasxtremely limited bioavailability of curcumin as only 22o 41g/mL was detectable in plasma when 8 g curcumin/day was given orally. Curcumin levels inhe microgramange have been showno beecessaryo show antiproliferativeffects in in vitro studies. Therefore, it was suggestedo heatsolubilize curcumin before administrationo increase its water solubility [147]. Moreover, bioactive compounds of curcumin degradation such as ferulic acid and vanillin also possess strong anticancer properties and can inhibit COX-1, COX-2 and significantly suppress NFκB activation [148][149][150].nhis wayhey may contributeohe observed biological activities of curcumin. Awaited areesults of ongoing clinicalrials on improved formulations of curcuminonhance chemo-oradiotherapy (see Table 2). There is a strongeed for more studies on differentatural compounds as growingvidence ismerging forheir benefits in improvingesults of standard anticancerreatments.

CONCLUSIONS
The power ofatural products lies in usinghem as adjuvantso standard anticancerherapies buthe struggle ishathey oftenxhibit contrary actions, depending on concentration. At high doses ( > 50 µM)atural compounds presented inhis article have pro-oxidant properties by limiting antioxidant capacity of cancer cells (Figure 3). Direct inhibition of cellular antioxidants or suppression of pathways leadingoheirxpression can sensitize cancer cellso chemo-andadiotherapy. Normal cells areothat sensitiveohe manipulations inedox homeostasis asheir growth and proliferation areothat much ROS-dependent. Contrarily, cancer cells operate under constant oxidative stress and are very sensitiveohe disruption ofheirnhanced abilityo scavenge freeadicals. Therefore, impairing antioxidant capacity ofumorsmerges as a good strategyoargethem. Especially inhibition of Nrf2 pathway seems a very promising approach as Nrf2 controlsxpression of crucial cellular antioxidants, drugfflux pumps and detoxificationnzymes. Simultaneous inhibition of Nrf2 and prosurvival NFĸB signaling isven moreffective in promoting death ofumor cells. Therefore,atural productshat suppress Nrf2 and NFĸB pathways are promising candidates for adjuvantso chemo-andadiotherapy allowing for loweringheir doses.t iseverthelessssentialo bear in mindhatheffecthey induce in cells depends onhe applied dose, cellype,xposureime andnvironmental conditions. The sameatural product in different concentrations often possesses contrary properties. This is why it is so challengingoranslateesults from in vitro modelso in vivo conditions. Concentrations used in cell linesxperiments are often very hardo achieve in patients. Givenhe poor plasmatic bioavailability of active compounds and biotransformation processeshey undergo inhe body,he circulating concentration ofatural compounds administered orally areather low. Moreover,he biologicalffectshey produce dootecessarilyeedo be a consequence ofhe action of onlyhe parent compound, but might also be assignedo its metabolites. Therefore,heffectsatural products present in vivo might be different orven oppositeoxpected and instead of potentiatingheffect of chemo-oradiotherapy,hey might weakenheir action. The majority of clinicalrialsest ROS scavenging properties ofatural compounds inhe context of cancer chemoprevention orheir abilityo alleviate sideffects of chemo-andadiotherapy. Just a few addressed a question of synergisticffects ofatural products with classic anticancerherapies andheesults so far warrant further investigation. There is a strongeed for clinical studiesestinghese combinationreatments in defined cancerypes with special focus on bioavailability and stability ofatural products.