Flavopiridol's antiproliferative effects in glioblastoma multiforme.

ORIGINAL ARTICLE

Year : 2016 | Volume : 12 | Issue : 2 | Page : 811--817

Flavop r dol's ant prol ferat ve effects n gl oblastoma mult forme

1 _{Department of Med cal B ology, Faculty of Med c ne, Gaz Un vers ty, Ankara, Turkey} 2 _{Department of Med cal B ology, Faculty of Med c ne, Ufuk Un vers ty, Ankara, Turkey}

Correspondence Address: Irem Dogan Turacl

Department of Med cal B ology, Faculty of Med c ne, Ufuk Un vers ty, Ankara Turkey

Abstract

A m of Study: Gl oblastoma mult forme (GBM) s largely refractory to surg cal operat on, rad otherapy, and chemotherapy n use today. Rema n ng l fet me account ng for the GBM-affected pat ents var es between 12 and 16 months generally. The most frequently altered genes n GBM are p53, ep dermal growth factor receptor, PTEN, and cycl n-dependent k nase nh b tor 2A. Our a m s to nvest gate the ant prol ferat ve and apoptot c effects of flavop r dol, a cycl n-dependent k nases and spec f c phosphok nase nh b tor, on gl oblastoma cell l nes hav ng d fferent genet c prof les: U87MG, U118MG, and T98G. Mater als and Methods: Cell v ab l ty and IC₅₀ values were detected by 3-(4,5-d methylth azol-2-yl)-2,5-d phenyltetrazol um brom de assay, prote n express ons were determ ned by Western blot and caspase act v t es were analyzed by act v ty k t. Results: Western blot analys s showed down-regulat on of the cycl n D1, c-Myc, and p53 prote n act v t es, and up-regulat on of p27KIP1 act v ty after flavop r dol treatment. Add t onally, flavop r dol d m n shed p-Akt prote n levels generally wh ch nduces nh b t on of prol ferat on. Conclus on: The present study demonstrated that flavop r dol d d not nduce caspase-3/7 act vat on, BIM, and BAX pro-apoptot c prote ns but t leads to the express on changes of var ous prote ns that nh b t prol ferat on and etern ty n gl oblastoma cell l nes wh ch have d fferent genet c alterat ons.

How to c te th s art cle:

Cobanoglu G, Turacl ID, Ozkan AC, Ekmekc A. Flavop r dol's ant prol ferat ve effects n gl oblastoma mult forme.J Can Res Ther 2016;12:811-817

How to c te th s URL:

Cobanoglu G, Turacl ID, Ozkan AC, Ekmekc A. Flavop r dol's ant prol ferat ve effects n gl oblastoma mult forme. J Can Res Ther [ser al onl ne] 2016 [c ted 2018 Jul 27 ];12:811-817

Ava lable from: http://www.cancerjournal.net/text.asp?2016/12/2/811/172132

Full Text

Introduct on

subgroups as astrocytoma Grades I, II, III, and IV, ol godendrogl omas, ependymomas, and m xed gl omas. Grade IV astrocytoma s known as gl oblastoma mult forme (GBM), wh ch s the most aggress ve and common form of gl oma.[1],[2] There are several genet c alterat ons assoc ated w th GBM format on and progress on such as mutat ons, ampl f cat ons, ep genet c, and gene express on changes. The common genet c alterat ons nclude ep dermal growth factor receptor (EGFR) ampl f cat on, INK4a-ARF loss, RB loss, p53 mutat ons, MET ampl f cat on, PIK3CA mutat ons, ERBB2 mutat on, PDGFR overexpress on, cycl n-dependent k nase 4 (CDK4) and CDK6 ampl f cat ons.[3],[4],[5],[6] One of the well-character zed mutat ons n the EGFR gene generates a truncated EGFR prote n known as EGFRvIII, wh ch lacks exons 2–7 of the EGFR gene.[7] Also, ampl f cat on or overexpress on of EGFR s a hallmark of GBM.[8] EGFR ampl f cat on s assoc ated w th delet on of the cycl n-dependent k nase nh b tor 2A (CDKN2A) gene locus.[9]

The PI3K/Akt pathway s usually act vated n gl oblastoma cells. Growth factor receptor mutat ons (EGFRvIII), PTEN, and PIK3CA mutat ons, Akt act vat on nduce these pathway alterat ons.[10] Phosphorylat on of Akt at S473 and T308 s requ red for full act vat on of Akt.[11],[12] When Akt s act vated, t nduces act vat on of several prote ns that play key roles n cell prol ferat on, apoptos s, and metabol sm. PRAS40, FOXOs, MDM2, p21, p27, BAD, NF-κb, caspase-9, TSC2 are a few examples of Akt substrates.[13]

Cell cycle control s another ant tumor gen c approach n gl oblastoma therapy. p53 tumor suppressor prote n has p votal funct ons n the s gnal ng pathways requ red to med ate cell cycle and apoptos s. It nteracts w th the promoters of p21, BAX, and many regulatory genes that have roles on apoptos s and cell cycle.[14],[15] Among the cell cycle prote ns, the cycl ns, CDKs, and CDK nh b tors have roles n gl al transformat on.[16] In response to st mulat ng s gnal ng from upstream pathways, cycl n D/CDK4, cycl n D/CDK6, and cycl n E/CDK2 complexes sequent ally coord nate to phosphorylate and nact vate RB. These phosphorylat ons d srupt RB-E2F cooperat on lead ng to nduct on of E2F-regulated gene express on and cell prol ferat on.[17] Due to RB repress on, the cell progresses nto S-phase. The act v ty of CDKs s suppressed by CDK nh b tors such as CIP/KIP (p21WAF1/CIP1, p27KIP1, and p57KIP2) and INK4 fam ly (p15INK4B, p16INK4A, p18INK4C, and p19INK4D).[16] So, CDK nh b tor pathway act vat on m ght be a prom s ng strategy to mprove eff cacy of cancer treatments n GBM.

Flavop r dol s a sem synthet c flavono d obta ned from dysoxylum b nectar ferum.[18] Flavop r dol nh b ts n v tro cell growth through CDKs (CDK2, CDK4, and CDK6) n G1/S or G2/M of cell cycle.[19],[20] Flavop r dol has been shown to delay tumor growth and cell m grat on and t depletes cycl n D1, CDK4, and Bcl-2 express on n GL261 mur ne gl oma cells. Also, flavop r dol treatment nduces caspase-dependent and ndependent apoptos s pathways. Wh le flavop r dol nduces caspase and cytochrome c ndependent pathway n human gl oma cells, t s m tochondr al med ated n mur ne gl oma cells.[21],[22] Also, flavop r dol plays an mportant role n the progress on to treatment res stance by reduc ng JUNB gene express on n mult ple breast cancer cell l nes.[23]

The genet c alterat ons on const tut vely act vated/ nact vated s gnal transduct on pathways such as p16, p53, PTEN, EGFR, and the r aberrant prote n express ons can be rat onal targets for GBM. Desp te several agents that are developed for targeted therap es, tumors got res stance to current therapeut cs. Today, alkylat ng agent temozolom de s used for the treatment of GBM after tumor resect on and rad otherapy. However, med an surv val s st ll l m ted to 15 months.[24] Flavop r dol, nh b tor of broad-spectrum CDKs, causes apoptos s and cell cycle arrest at the G1/S and G2/M boundar es. In th s study, we nvest gated the ant prol ferat ve and apoptot c effects of flavop r dol on GBM cell l nes based on the r genet c background [Table 1].{Table 1}

Mater als and Methods

Cells and reagents

All the cell l nes used n th s study (U87MG, T98G, and U118MG) were grown n DMEM (Lonza) supplemented w th 5% heat-nact vated fetal bov ne serum (Lonza), 100 U/ml pen c ll n and 100 μg/ml streptomyc n (Lonza). Cells were grown n a hum d f ed ncubator conta n ng 5% CO2 and 95% a r at 37°C.

Flavop r dol was obta ned from Enzo. A 10 mM stock solut on was prepared n d methyl sulfox de (DMSO). Flavop r dol (150 nM-10 µM) was d luted and added to cells that were grow ng on cell culture d shes. All assays were performed at 24, 48, and 72 h of ncubat on.

Cell v ab l ty

Cell v ab l ty was determ ned by 3-(4,5-d methylth azol- 2-yl)-2,5-d phenyltetrazol um brom de (MTT) assay. Cells were seeded n DMEM at 7 × 103 cells/200 µl per well n 96-well plates for 24 h at 37°C. When cells were attached after 24 h, cells were treated w th DMSO (control group) and flavop r dol (156 nM; 312 nM; 625 nM; 1.25 µM, 2.5 µM, 5 µM, and 10 µM) for 24, 48, and 72 h per ods. After ncubat on t me, 10 µl MTT solut on (5 mg/ml) was added to each well. After 4 h of MTT ncubat on at

37°C, 100 µl crystal d ssolv ng buffer was added and the plates were gently shaked on an orb tal shaker for 5 m n. The absorbance at 570 nm was measured w th a m croplate reader. Each treatment was repeated 4 t mes. The mean absorbance of four wells was used as an nd cator of relat ve cell growth.

Immunoblott ng

Cells were plated at a dens ty of 5 × 105 per well n s x-well plates. The follow ng day, cells were treated w th drug or equal volume of DMSO for 6, 24, and 48 h. Cell extracts were prepared by add ng 2x Laemml sample buffer supplemented w th a phosphatase and protease nh b tor cockta l. Lysates were son cated and the prote n concentrat on was quant f ed us ng b c nchon n c ac d (BCA) k t (Thermo, MA, USA). Equ valent prote n was loaded and prote ns were separated by sod um dodecyl sulfate polyacrylam de gel electrophores s then transferred to 0.45 µm n trocellulose membranes. Equ valent load ng was conf rmed by sta n ng membranes w th Ponceau S dye. Membranes were blocked for 1 h n block ng buffer (5% m lk, 1× TBS, 0.1% Tween 20) and placed n pr mary ant body (5% bov ne serum album n, 1× TBS, 0.1% Tween 20) overn ght at 4°C. The follow ng day, membranes were washed thr ce n wash buffer (1× TBS, 0.1% Tween 20). Pr mary ant bod es aga nst Akt, p-Akt, p53, p-p53, EGFR, p-EGFR, p27, BIM, BAX, cycl n D1, c-Myc, GAPDH, secondary ant -rabb t IgG, and ant -mouse IgG were purchased from Cell S gnal ng Technolog es (Danvers, MA, USA). Prote ns were detected us ng horserad sh perox dase-l nked secondary ant bod es and v suadase-l zed w th the enhanced chem dase-lum nescent detect on system (GE Headase-lthcare B osc ences, P ttsburgh, PA, USA). Immunoblot exper ments were performed at least tw ce.

Caspase 3/7 prote n act v ty

About 1× 106 cells were seeded n 6-well plates and ncubated w th DMSO (control group) and flavop r dol for 6, 24, and 48 h. Cell extracts were prepared by add ng 2× Laemml sample buffer supplemented w th a phosphatase and protease nh b tor cockta l. Lysates were son cated and prote n concentrat on of each sample was determ ned by BCA k t (Thermo, MA, USA). Caspase 3/7 prote n act v ty was measured by “AnaSpec Sensolyte Homogeneous AFC Caspase 3/7 Assay K t” (AnaSpec, CA, USA) accord ng to the assay protocol.

Stat st cal analys s

D fferences n cytotox c ty and prote n act v ty levels were analyzed us ng S gmaStat software (vers on 12.3) by Systat Software, Inc. by us ng Student's t-test. P < 0.05 was cons dered stat st cally s gn f cant.

Results

Ant prol ferat ve effects of flavop r dol (10 µM, 5 µM, 2.5 µM, 1.25 µM, 625 nM, 312 nM, 156 nM) was tested on T98G (mt-p53), U118MG (mt-p53), and U87MG (wt-p53) cells at 24, 48 and 72 h per ods, respect vely. For U118MG cells, although flavop r dol decreased cell v ab l ty even at 0.156 µM at 72 h, ts IC50 value was seen at 0.6 µM at 48 h. Also, there was no s gn f cant death rate at the h ghest concentrat on (10 µM) at 24 h [F gure 1]a. Although t was not s gn f cant at 0.156, 0.312, 0.625, and 2.5 µM, the prol ferat on rate was decreased accord ng to ncreased flavop r dol doses at T98G cells at 24 h. The IC50 value was determ ned around 300 nM at 48 h for T98G cells and all appl ed doses of flavop r dol decreased cell v ab l ty s gn f cantly at both 48 and 72 h per ods [F gure 1]b. For U87MG cells, flavop r dol decreased prol ferat on s gn f cantly at 300 nM and h gher doses at all-t me po nts stud ed. The IC50 value was determ ned around 300 nM at 48 h for U87MG cells [F gure 1]c. {F gure 1}

To nvest gate the prote n express on changes between control and flavop r dol treated groups, Western blot analys s was performed for several prol ferat ve and apoptot c prote ns at 6, 24, and 48 h. Western blot analys s showed that wh le 600 nM flavop r dol decreased p-Akt, c-Myc, and cycl n D1 express on n a t me-dependent manner, t ncreased p27KIP1 express on at all-t me po nts at U118MG cells. However, p-p53 express on was not orderly changed w th flavop r dol treatment. Also, the pro-apoptot c marker express ons were not coord nated w th each other, whereas BAX express on was ncreased by flavop r dol appl cat on at 24 and 48 h, BIM express on was decreased at all t me po nts at U118MG cells [F gure 2].{F gure 2}

For T98G cells, although p-Akt s gnal was decreased at 6 h, t was ncreased by 300 nM flavop r dol appl cat on at 24 and 48 h. Also, cycl n D1 express on was only decreased by flavop r dol at 48 h, where c-Myc express on was decreased at all t me po nts. Wh le p-p53 s gnal was ncreased at all t me po nts, p27 prote n express ons were ncreased w th flavop r dol treatment at 24 and 48 h. Interest ngly, BAX pro-apoptot c prote n express on was not changed by flavop r dol, where BIM express on was decreased at 24 and 48 h [F gure 2].

Treatment w th 300 nM flavop r dol reduced p-Akt s gnal only at 48 h at U87MG cells. Also, mportant cell cycle prote n cycl n D1 and c-Myc express on was decreased by flavop r dol treatment at all t me po nts. Parallel to these results, p-p53 s gnal was nduced w th flavop r dol appl cat on. On the other hand, p27 prote n express on was decreased as the oppos te of p-p53 s gnal. Also, BIM and BAX pro-apoptot c prote n express ons were reduced by flavop r dol treatment at U87MG cells [F gure 2].

When the GBM cells used n th s study were treated w th flavop r dol (T98G (300 nM), U118MG (600 nM) and U87MG (300 nM)) at 6, 24 and 48 h, the caspase 3/7 act v ty was not changed s gn f cantly [F gure 3]a, [F gure 3]b, [F gure 3]c. Our results nd cate that flavop r dol nduced down-regulat on of mportant regulators n cell cycle and prol ferat on n all cell l nes ndependent of genet c status.{F gure 3}

D scuss on

Understand ng the mutat ons, cell cycle regulat on and cell death w ll be benef c al for dent fy ng the pathogenes s and the appropr ate treatment of GBM. PTEN, EGFR, p53 mutat ons, and loss of chromosome 9p, wh ch codes for CDKN2A and CDKN2B genes, are common genet c alterat ons n GBM. CDKN2A locus has two overlapp ng genes p14ARF and p16INK4a, wh ch are ent rely d fferent tumor suppressor prote ns.[25],[26] Wh le p16INK4a regulates RB act vat on, p14ARF regulates p53 pathway. P16INK4a b nds and nh b ts cycl n D/CDK4/6 complex thereby prevent ng RB phosphorylat on and causes G1/S cell cycle arrest.[27] Thus, the loss of p16INK4a and/or p14ARF wh ch s mportant for GBM format on and treatment d rectly affects cell cycle control.[28] Flavop r dol s a CDK nh b tor that can be used to nh b t cell cycle progress on. It has been used n cl n cal tr als e ther as a s ngle agent or n comb nat on w th other agents.[29],[30] Although flavop r dol s known to nh b t CDKs, t st ll has to be clar f ed how flavop r dol causes apoptos s.

In our study, we treated the GBM cell l nes w th var ous doses of flavop r dol for 24, 48, and 72 h and got IC50 values at 300 nM for U87MG and T98G, and at 600 nM for U118MG. However, Brüsselbach et al. reported IC50 concentrat ons were 144 nM for A549 nonsmall cell lung cancer (NSCLC) cells, 16 nM for LNCaP, 94 nM for HuVEC cells and 154 nM for LoVo cells. [31] On the other hand, for A549 cells, 200–500 nM cont nuous flavop r dol resulted n reduced cell growth at 72 h.[32] IC50 value of flavop r dol was 500 nM for CD133+/CD44+ human prostate cancer stem cells.[33] Even the IC50 values of flavop r dol change accord ng to cell type, n parallel w th our study, Alonso et al. found flavop r dol nh b ted cell prol ferat on at 300 nM concentrat on at gl oma cell l nes ndependent of p53 status.[21]

There are d st nct ve stud es demonstrat ng how flavop r dol leads to apoptos s n var ous cell l nes. Although the tumor suppressor gene p53 s requ red for the eff c ent act vat on of apoptos s and p-p53 levels were upregulated by flavop r dol n T98G and U87MG cells n our study. Shap ro et al. suggested flavop r dol-med ated apoptos s was ndependent of p53 by us ng p53 w ld type A549 lung carc noma cells.[32] Alonso et al. showed flavop r dol decreased XIAP, Bcl-XL, p21, MDM2, IAP2, and surv v n mRNA levels and nduces apoptos s ndependent of p53 and caspase-related mechan sm n gl oma cells. [21] Also, overexpress on of MDM2 resulted n res stance of p53 w ld type U87MG cells to c splat n- nduced apoptos s but d d not change p53 express on.[34] Dem denko and Blagosklonny showed low doses of flavop r dol nduces p53 by nh b t ng MDM2 and p21 at HCT116 cells and also promotes apoptos s n p53 null cells.[35] So, t st ll rema ns unclear how p53 jo ned flavop r dol's ant tumor gen c act v t es n our and other stud es.

In parallel w th Alonso et al., the caspase 3/7 levels were not changed s gn f cantly by flavop r dol treatment n GBM cells n our study. However, Takada et al. showed flavop r dol suppressed tumor necros s factor (TNF)- nduced act vat on of Akt, c-Myc, Bcl-2, Bcl-XL, and several ant -apoptot c prote ns. Moreover, they showed TNF-related apoptos s depends on caspase act vat on n human myelo d cells.[36] Flavop r dol- nduced growth nh b t on and apoptos s at 500 nM results n a s gn f cant ncrease n mmunofluorescence sta n ng of caspase-3, caspase-8, and p53 n human prostate cancer stem cells.[33] Th s d screpancy on dependence on caspase act vat on may be due to cancer stem cell dynam cs or d fferent cancer cell types. Also, treatment of mult ple myeloma cell l nes w th 200 nM flavop r dol nduced apoptot c cell death but decreased ant -apoptot c Mcl-1 prote n and mRNA express on.[37] Also, 200 nM flavop r dol treatment resulted n evaluat on of E2F1 prote n wh ch represses Mcl-1 and nduce apoptos s n H1299 lung carc noma cell l ne.[38]

PI3K-Akt s gnal ng pathway nh b ts apoptos s by regulat ng several cell surv val molecules. In cerebellar granule neurons, flavop r dol has shown to be a neuroprotect ve agent n such a manner that t nh b ted GSK3b and Akt and prevent apoptos s wh ch s nduced by a PI3K nh b tor.[39] Th s f nd ng has mportant mpl cat ons on chemores stance mechan sms n GBM. Also, Akt phosphorylat on was decreased n a t me-dependent manner w th flavop r dol treatment n U118MG and U87MG cells n our study. It could be nterpreted as flavop r dol has ant prol ferat ve and k nase nh b t ng effects on PI3K and PDK1 k nases. BAX and BIM prote ns are the markers wh ch can nduce apoptos s n cancer cells. However, we could not observe ncreased BAX prote n express on w th flavop r dol treatment. S m lar to our study, n B-cell chron c lymphoblast c leukem a cells, flavop r dol adm n strat on nh b ted Bcl-2 and Mcl-1 express on, but t d d not nduce BAX express on.[40] On the other hand, Pe et al. showed flavop r dol and ts comb nat on w th Bcl-2 nh b tor HA14-1 tr ggered m tochondr al damage and lead to m tochondr al translocat on of BAX n human myeloma cells.[41] Also, BAX was upregulated and cycl n D1 was downregulated by flavop r dol n Eca109 esophageal cancer cells.[42] In l ne w th prev ous work n w ld type and mutant p53 gl oma cell l nes, Alonso et al. observed Bcl-2 prote n express on was downregulated whereas BAX prote n levels were upregulated at 24 and 48 h, result ng n a low Bcl-2/BAX rat o, wh ch s an nd cator of apoptos s.[21] We could not observe BIM prote n upregulat on w th flavop r dol adm n strat on n our study. However, comb nat on of flavop r dol and obatoclax, a BH3-m met c, nh b ted Mcl-1 and tr ggered BIM transcr pt on and nduce apoptos s n human myeloma cells.[43]

Although we could not see apoptot c st mulus n our study, prol ferat on markers were downregulated by flavop r dol appl cat on n gl oblastoma cell l nes. In parallel w th our study, Alonso et al. showed cycl n D1 levels decreased after flavop r dol treatment. By decreas ng cycl n D1 prote n levels, CDK4 k nase act v ty s decreased, wh ch results n the Rb hypophosphorylat on that results n growth arrest at G1/S-phase.[21] Also, t has been reported flavop r dol decreased cycl n D1 express on n MCF-7 cells.[44] At the same t me, c-Myc express on was nh b ted by flavop r dol n all cell l nes n our study. The decrease n c-Myc express on may be due to nh b t on of cycl ns and CDKs by flavop r dol n GBM cells. S m lar to our f nd ngs, flavop r dol suppressed TNF- nduced c-Myc express on n human myelo d cells.[36]

p27KIP1, a CDK nh b tor and a negat ve regulator of cell cycle, was upregulated by flavop r dol n p53 mutant U118MG and T98G cells n our study. However, we observed t was upregulated by flavop r dol n p53 w ld-type U87MG cells. Also, Shap ro et al. showed flavop r dol- nduced p27KIP1 cleavage n p53 w ld type A549 NSCLC cells, wh ch may be a consequence of apoptos s.[32] In B cell chron c lymphocyt c leukem a cells, flavop r dol downregulated p27KIP1 expresss on.[45] Lechpammer et al. showed N-methyl-N-benzyln trosam ne and gastroduodenal-esophageal reflux nduces Barrett's esophagus and mal gnant transformat on of the esophageal mucosa n p27 knockout m ce. Flavop r dol treatment reduced cycl n D1 express on and Barrett's esophagus format on n those m ce.[46] In uter ne le omyoma cells, flavop r dol has been used as chemoprevent ve and therapeut c agent that nduced G1 cell cycle arrest and ncreased p27KIP1 and p21 express ons.[47] In summary, the present study focused on the ant prol ferat ve and apoptot c effects of flavop r dol on GBM cell l nes based on the r genet c background. The express on prof les of our apoptot c markers showed no s gn of flavop r dol- nduced apoptos s. Although we could not observe apoptot c st mulus, the prol ferat on rates were decreased w th flavop r dol treatment n all cell l nes used n th s study regardless of the r genet c makeup. It s mportant to emphas ze that these results probably nd cates other pathways or apoptot c markers that we d d not search are changed by flavop r dol appl cat on. Further n v vo stud es are necessary to clar fy the poss ble mechan sms and other s gnals to solve flavop r dol's effects on cell cycle and apoptos s. Acknowledgment

Th s study was supported by the Gaz Un vers ty Research Fund as a research project w th code number 01/2010-13. F nanc al support and sponsorsh p

N l.

Confl cts of nterest

There are no confl cts of nterest.

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