T-Type Ca þ Channel Inhibition Induces p53-Dependent Cell ...Cell Death and Survival T-Type Ca2þ Channel Inhibition Induces p53-Dependent Cell Growth Arrest and Apoptosis through

Cell Death and Survival

T-Type Ca2þ Channel Inhibition Induces p53-DependentCell Growth Arrest and Apoptosis through Activation ofp38-MAPK in Colon Cancer Cells

Barbara Dziegielewska1, David L. Brautigan2,3, James M. Larner1,3, and Jaroslaw Dziegielewski1,3

AbstractEpithelial tumor cells express T-type Ca2þ channels, which are thought to promote cell proliferation. This study

investigated the cellular response to T-type Ca2þ channel inhibition either by small-molecule antagonists or byRNAi-mediated knockdown. Selective T-type Ca2þ channel antagonists caused growth inhibition and apoptosismore effectively in HCT116 cells expressing wild-type p53 (p53wt), than in HCT116mutant p53�/� cells. Theseantagonists increased p53-dependent gene expression and increased genomic occupancy of p53 at specific targetsequences. The knockdown of a single T-type Ca2þ channel subunit (CACNA1G) reduced cell growth andinduced caspase-3/7 activation in HCT116 p53wt cells as compared with HCT116 mutant p53�/� cells.Moreover, CaCo2 cells that do not express functional p53 were made more sensitive to CACNA1G knockdownwhen p53wt was stably expressed. Upon T-type Ca2þ channel inhibition, p38-MAPK promoted phosphorylationat Ser392 of p53wt. Cells treated with the inhibitor SB203580 or specific RNAi targeting p38-MAPKa/b(MAPK14/MAPK11) showed resistance to T-type Ca2þ channel inhibition. Finally, the decreased sensitivity tochannel inhibitionwas associated with decreased accumulation of p53 and decreased expression of p53 target genes,p21Cip1 (CDKN1A) and BCL2-binding component 3 (BBC3/PUMA).

Implications: A novel pathway involving p53 and p38-MAPK is revealed and provides a rationale for antitumortherapies that target T-type Ca2þ channels. Mol Cancer Res; 12(3); 348–58. �2013 AACR.

IntroductionIntracellular Ca2þ regulates many cellular processes,

including cell cycle, proliferation, transcription, exocytosis,hormone release, cell motility, and apoptosis (1, 2). Voltagegated Ca2þ channels facilitate transient Ca2þ influx fromthe environment into the cytoplasm and appear mostly inexcitable tissues, but also are unusually expressed in cancercells. Low-voltage activated Ca2þ channels, termed T-typeCa2þ channels, recently gained attention in cancer therapy,because their inhibition decreased proliferation of glioblas-toma cells (3, 4), breast adenocarcinoma cells (5, 6), mel-anoma cells (7), and esophageal carcinoma cells (8). Inaddition, an antagonist selective for T-type Ca2þ channels,mibefradil, has been proposed recently as a sensitizing agent

with activity in vivo in combination with chemo- (9) orradiotherapy (10). Aberrant expression of T-type Ca2þ

channels in cancer cells is thought to promote cell survival,proliferation, and motility (3, 11); however, the molecularmechanisms for these effects are poorly understood.Signaling through voltage gated Ca2þ channels involves

Ca2þ-binding proteins such as calmodulin (CaM) andactivation of its binding partner Ca2þ/calmodulin–depen-dent kinase II (CaMKII), which in turn regulates T-typeCa2þ channels activity (12, 13). Consequently, proteinsdownstream from activated CaMKII, such as mitogen-acti-vated protein 3 kinase 5 (MAP3K5, also known as apoptosissignal–regulating kinase 1 or ASK-1) or prosurvival proteinkinase B (PKB also known as AKT) are affected by changes inintracellular Ca2þ concentration (14–16). Increased CaM-KII activity regulates gene expression directly through phos-phorylation of transcription factors, such as cyclic-AMPresponse element binding protein (CREB; reviewed inref. 17) or indirectly involving p53 activation (18, 19). Forexample, CREB transcription factor could be responsible forradiation resistance through regulation of DNA repair genes(20), whereas intracellular Ca2þ and CaMKII could regulateefficient p53 activation upon 5-fluorouracil treatment thatinvolves activated p38-MAPK (19).Under physiologic conditions the levels of p53 protein

and its activity are low, but exposing cells to stress resultsin p53 induction and activation (21). Activated p53

Authors' Affiliations: Departments of 1Radiation Oncology, 2Microbiol-ogy, Immunology, andCancer Biology, Center for Cell Signaling, Universityof Virginia School of Medicine; and 3Cancer Center, University of Virginia,Charlottesville, Virginia

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Jaroslaw Dziegielewski, Department of RadiationOncology, University of Virginia School of Medicine, P.O. Box 800383,Charlottesville, VA 22908. Phone: 434-982-0076; Fax: 434-243-9789;E-mail: [email protected]

doi: 10.1158/1541-7786.MCR-13-0485

�2013 American Association for Cancer Research.

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accumulates as a tetramer in the cell nucleus and acts as atranscription factor, mediating expression of cell cycle-reg-ulating, senescence-inducing, and proapoptotic genes (22).Previously, mibefradil was shown to decrease proliferation ofesophageal carcinoma cells by increasing transcription of thecyclin-dependent kinase (CDK) inhibitor p21Cip1/Waf1(8). This suggested to us that cellular responses to T-typeCa2þ channels might be p53 dependent.In general, p53 is phosphorylated by protein kinases of

the phosphatidylinositol 3-kinase-related kinase familythat are activated by DNA damage, such as ataxia telan-giectasia mutated (ATM), ataxia telangiectasia mutatedand Rad3 related (ATR), and DNA-dependent proteinkinase (DNAPK). Phosphorylation releases p53 from itsinteractions with MDM2 ubiquitin ligase, which stabilizesp53, and allows for its specific DNA binding (23, 24).Following DNA damage induced by ionizing radiation orUV radiation, at least 17 Ser/Thr residues are phosphor-ylated within p53. Significant redundancies are observedas a single residue can be subject to phosphorylation bymultiple protein kinases, for example, Ser15-p53 is report-edly phosphorylated by ATM, ATR, DNAPK, p38-MAPK, and ERK (extracellular signal–regulated kinase)kinases (reviewed in ref. 21). Interestingly, some of theprotein kinases that interact and phosphorylate p53belong to the calmodulin-dependent kinase super family(which includes Chk1, Chk2, and death associated proteinkinase 1 and 3; ref. 25), and these may phosphorylate p53in response to pathologic Ca2þ signaling.Cross-talk is apparent between T-type Ca2þ channel

signaling and mitogen-activated protein kinase (MAPK)pathways with consequences for p53. Mobilization ofCa2þ through T-type Ca2þ channels (Cav 3.1; CACNA1G)produces a transient decrease in activity of Raf/MEK/ERK(26). Although ERK tends to induce prosurvival pathways,stress-activated MAPKs, such as p38-MAPK and JNK (c-jun–NH2–kinase), are linked to induction of apoptosis (27,28). In particular, upon UV-induced DNA damage orosmotic shock, p38-MAPK phosphorylates p53 at Ser46(29) or Ser33 (30), thus stabilizing p53. Moreover, UV-induced DNA damage activates p38-MAPK to activatecasein kinase 2 (CK2), which in turn also phosphorylatesp53 at Ser392, increasing p53 transcriptional activity (31–33). Thus, p38-MAPK supports increased p53 levels andactivity.In this study, we evaluated the actions of T-type Ca2þ

channels using two isogenic epithelial colon carcinoma celllines, HCT116 p53 wild-type (p53wt) and the HCT116p53-deleted counterpart (HCT116 p53�/�). In addition,we compared CaCo2 cells that do not express detectable p53protein due to a nonsensemutation in exon 6 of the p53 gene(Glu to Stop codon; ref. 34) with cells rescued for p53 bystable transfection with either vector (pcDNA3.1) or p53wtgene (pcDNA3.1 p53wt). We uncovered a relationshipbetween T-type Ca2þ channel inhibition and p53-depen-dent growth inhibition and apoptosis that implicate a p38-MAPK kinase signaling circuit. Our results support therationale for future development of T-type Ca2þ channel

blockers as chemotherapeutic agents that can induce cancercell death.

Materials and MethodsCell culture and drug treatmentColon carcinoma HCT116 p53wt and p53-deleted

mutant were described previously (35) and were maintainedin McCoy 5A medium (Life Technologies) supplementedwith 10% FBS (Life Technologies). CaCo2 colon adeno-carcinoma cell line was purchased from American TypeCulture Collection and cultured in Minimum EssentialMedium (MEM) supplemented with L-glutamine, nones-sential amino acids, and 20% FBS (Life Technologies). Thecell lines were not authenticated. Mibefradil and TTL1177were generously provided by Tau Therapeutics LLC.NNC55–0396 hydrate was from Sigma-Aldrich. Thep38-MAPK inhibitor SB203580 was from Santa CruzBiotechnology. All inhibitors were prepared in dimethylsulfoxide (DMSO; 10 mmol/L stock) and diluted in mediabefore the use.

Drug-induced growth inhibitionHCT116 cells were seeded in 96-well plates at 1,000 cells

per well. Twenty-four hours later, cells were treated intriplicates with different concentrations of T-type Ca2þ

channels inhibitors (mibefradil or TTL1177). After contin-uous exposure to drug for 4 days, cells were fixed withtrichloroacetic acid (10%) and stained with sulforhodamineB solution (0.1%; ref. 36). Percentage of growth inhibitionwas determined after background subtraction based on thecomparison of the absorbance signal of drug-treated cellsversus the control.

Plasmid DNA and siRNA transfection and growthinhibitionCaCo2 cells were transfected with pcDNA3.1 p53wt or

pcDNA3.1 empty vector plasmids [kind gift from Dr. A.Dutta, University of Virginia (UVA), Charlottesville, VA]using lipofectamine LTX and plus reagent (Life Technolo-gies) according to the manufacturer's instructions. Colonieswere selected for 8 weeks in media containing selectionantibiotic geneticin G418 (600 mg/mL; Life Technologies).In RNAi experiments the cells were transfected with 25nmol/L of siRNA [scrambled: 50-gacgaaagaccacucaauu/50-aauugaguggucuuucguc, siCACNA1G-ORF (#3166; 1):50-uuguagaggacuuuguucc/50-ggaacaaaguccucuacaa, siCAC-NA1G-ORF (#5356; 2): 50-auuuccuccagcgugaugc/50-gcau-cacgcuggaggaaau; all from Invitrogen/Life Technologies], orwith 10 nmol/L of siRNA pool of four different sequencesfor each gene (Santa Cruz Biotechnology) against MAPK14and MAPK11 (p38-MAPKa and p38-MAPKb, respective-ly), using lipofectamine RNAiMax (Life Technologies)according to the manufacturer's instructions. HCT116 orCaCo2 cells were seeded at 105 per well and transientlytransfected 24 hours later with appropriate siRNA con-structs. HCT116 cells were trypsinized after 72 to 96 hours,counted using trypan blue exclusion assay and collected by

p53 and p38-MAPK Response to T-Type Ca2þ Channels Inhibition

www.aacrjournals.org Mol Cancer Res; 12(3) March 2014 349




centrifugation for total RNA isolation. For CaCo2, cellswere reseeded after 24 hours at 500 cells per well into newdishes and allowed to form colonies for 14 days beforestaining with Coomassie Brilliant Blue R-250.

Caspase-3/7 activity assayCells seeded at 3 � 105 per well in 6-well plates were

transfected with 25 nmol/L of specific siRNA [scrambledcontrol or siCACNA1G (1)]. Twenty-four hours later, cellswere trypsinized, counted, and reseeded at 6,000 cells perwell into an opaque 96-well plate. Caspase-3/7 activity wasmeasured 48 hours posttransfection using the Apo-ONEcaspase activity assay kit according to the manufacturer'sinstructions (Promega). Caspase-3/7 activity was measuredin triplicates and represented as a fold-increase of fluores-cence calculated by comparing siRNA targeted cells withcells treated with scrambled siRNA.

Reverse transcriptase quantitative PCRTotal RNA was isolated using the RNeasy Kit (Qiagen)

and 1 mg was used for cDNA synthesis using the iScriptcDNA Synthesis Kit (Bio-Rad Laboratories) according tothe instructions. Each quantitative PCR (qPCR) reactionwas done in triplicate and included 50 ng of cDNA as atemplate, specific primers (Supplementary Table S1;ref. 37) and SsoFast EvaGreen master mix (Bio-RadLaboratories). Conditions for amplification were as fol-lows: initial denaturation 98�C for 30 seconds, followedby 40 cycles of denaturation for 5 seconds at 98�C, andannealing with extension for 5 seconds at 62�C. Relativegene expression of specific genes of T-type Ca2þ channelswas normalized on the basis of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level.Normalized gene expression was calculated by the formula2ð�DDCtÞ by subtracting the Ct value of GAPDH and thenthe Ct value of untreated control.

Western blottingHCT116 cells were seeded at 2.5� 105 in 35-mm plates

48 hours before mibefradil treatment for 9 or 24 hours. Aftertreatment cells were trypsinized, counted, and resuspendedat 1� 107 cells/mL in a modified RIPA buffer (20 mmol/LTris pH 7.6, 150 mmol/L NaCl, 5% glycerol, 1 mmol/LEDTA, 1% deoxycholate plus protease inhibitors, andphosphatase inhibitors; Sigma-Aldrich). Equivalent of30,000 cells per lane were resolved by electrophoresis inSDS–PAGE, transferred to the nitrocellulose membrane(0.2 mm; Bio-Rad Laboratories) and probed with specificantibodies anti-p53 (1:200; Santa Cruz Biotechnology),anti-CACNA1G (1:500; anti-Cav 3.1; Abcam), anti-CAC-NA1H (1:500; anti-Cav 3.2; cloneN55/10;UCDavis/NIHNeuroMab Facility), anti-phospho Ser392 p53 and anti-phospho Ser15 p53 (1:1,000; Cell Signaling Technology),anti-p21 and anti-PUMA (1:1,000; Cell Signaling Tech-nology), and anti-b-actin (1:10,000; Sigma-Aldrich). Theproteins of interest were visualized using a two-color Li-COR Odyssey Imager (LI-COR) and quantified usingImageJ software (38).

Chromatin immunoprecipitation assayChromatin immunoprecipitation (ChIP) assay was per-

formed according to the published protocol (39). Briefly,cells were seeded in 100-mm dishes at 2.5 � 106 2 daysbefore treatment with mibefradil at 10 mmol/L, or expo-sure to ionizing radiation (6 Gy). After 9 hours drugtreatment or 6 hours after irradiation, macromoleculeswere cross-linked with 1% formaldehyde for 10 minutes.Cells were lysed in buffer containing 1% SDS, proteaseinhibitors (Pierce; Thermo Fisher Scientific) and 100 mg/mL of sonicated herring sperm genomic DNA. Detergent-insoluble fractions were collected by centrifugation at14,000 rpm for 10 minutes at 4�C, resuspended in0.25% SDS, 0.25 mmol/L NaCl, protease inhibitors(Pierce), and 100 mg/mL herring sperm genomic DNA,and sonicated using Branson Sonifier 400 W Cell Dis-ruptor. Extracts were diluted with three volumes of 350mmol/L NaCl, 1% NP40 plus protease inhibitors, andspecific antibodies (mouse monoclonal anti-p53 or mousemonoclonal IgG; EMD Millipore) were added for over-night incubation. Protein–DNA complexes were collectedwith protein G sepharose beads (Sigma-Aldrich) andwashed four times with 350 mmol/L NaCl, 1% NP40,50 mg/mL of herring sperm genomic DNA. Complexeswere dissociated from beads, cross-links were reversed withheat (65�C/4 h), and DNA was phenol–chloroformextracted and precipitated with 0.7 volume of 100%isopropanol. Mibefradil or ionizing radiation inducedenrichment of p53 binding to specific sites was deter-mined on the basis of the qPCR reaction normalized to theinput and to the signal of untreated HCT116 p53wtcontrol according to the formula: 2ð�DDCtÞ. Specific pri-mers were designed using Primer3Plus software (40) andeither included or were in a close proximity to a specificbinding site for p53 according to previously publisheddata (Supplement Table S2; ref. 41).

Statistical analysesAll values are expressed as the means of at least three

independent experiments � SEM. Results were comparedusing one-factor ANOVA analysis. A P value of less than0.05 indicated statistically significant differences.

ResultsInhibition of T-type Ca2þ channels induces p53-dependent apoptosis in HCT116 cellsWe testedmibefradil and the structurally unrelatedT-type

Ca2þ channel blocker TTL1177 in HCT116 p53wt andp53�/� cells, a well-established model system to comparep53-dependent cellular responses (35). Cells were treatedwith increasing concentrations of mibefradil or TTL1177,or vehicle as control, for 96 hours and assayed for prolifer-ation as described in Materials and Methods. Both mibe-fradil and TTL1177 decreased growth of HCT116 cellsrelative to controls in a dose-dependent manner; however,the p53wt cells showed greater sensitivity than HCT116p53�/� cell line (3.3- and 3.1-fold difference in sensitivity tomibefradil and TTL; Fig. 1A and C, respectively).

Dziegielewska et al.

Mol Cancer Res; 12(3) March 2014 Molecular Cancer Research350




Preferential inhibition of HCT116 p53wt over p53�/� cellsalso was observed with NNC55–0396, an analog of mibe-fradil with greater selectivity for T-type Ca2þ channels (datanot shown; ref. 42). Notably, the reduction in cell prolif-eration in response to either mibefradil or TTL1177 wasaccompanied by a significant increase in activation of cas-pase-3/7 (Fig. 1B and D). Treatment of p53wt cells for 24hours with 10 mmol/L mibefradil resulted in a 9� 1.8–foldincrease in caspase-3/7 activity as compared with the vehicle-treated control, whereas TTL1177 gave a 2.9 � 0.1–foldincrease. Under the same conditions neither mibefradil norTTL1177 induced caspase activity in p53�/� HCT116cells. Thus, reduced proliferation and induction of apoptosisin response to blockade of T-type Ca2þ channels by differentagents seems to depend upon p53.

Knockdown of CACNA1G T-type Ca2þ channel inHCT116 cells results in a p53-dependent reduction ofcell proliferation and induction of apoptosisWe measured the relative expression of T-type Ca2þ

channels subunits CACNA1G (Cav3.1) and CACNA1H(Cav3.2) in HCT116 p53wt and p53�/� cells using reversetranscriptase quantitative PCR (RT-qPCR; Fig. 2A).HCT116 p53wt and p53�/� cells express mRNA for bothCACNA1G and CACNA1H subunits at comparativelysimilar levels. Consistent with similar mRNA levels, usingspecific antibodies, we observed the same levels of CAC-NA1G (Cav3.1) and CACNA1H (Cav3.2) proteins byimmunoblotting extracts of HCT116 p53wt and p53�/�

cells (Fig. 2A, inset).

To rule out the possibility that the observed effects ofmibefradil and TTL1177 on HCT116 cells were due to off-target actions rather than inhibition of T-type Ca2þ chan-nels, we used siRNA-mediated knockdown of CACNA1G.Cells were transiently transfected with two different siRNAstargeting CACNA1G, si(1) and si(2). The efficiency ofknockdown was approximately 75% to 80% as measuredby RT-qPCR (Fig. 2B). Reduction of CACNA1G expres-sion significantly decreased growth of HCT116 p53wt cells(Fig. 2C). Interestingly, depletion of CACNA1G alsoreduced the proliferation ofHCT116 p53�/� cells, althoughnot as extensively as in p53wt cells, suggesting that decreasedexpression of T-type Ca2þ channels can affect proliferationindependent of p53 status (Fig. 2C). Inhibition of cellproliferation induced by specific siRNA was accompaniedby a significant increase of caspase-3/7 activity in HCT116p53wt cells (2.4 � 0.15–fold increase over vehicle-treatedcells; Fig. 2D).

Expression of p53wt sensitizes CaCo2 cells to aknockdown of T-type Ca2þ channelsWe used CaCo2, another colon carcinoma cell line

previously reported to express T-type Ca2þ channels(43). CaCo2 are deficient in functional p53 due to anonsense mutation in exon 6 of the p53 gene (Glu to Stopcodon; ref. 34); thus, for a pairwise comparison, weestablished cell lines stably transfected with the p53wtgene. The expression of mRNA for T-type Ca2þ channelssubunits CACNA1G and CACNA1H (Fig. 3A) and p53protein level (Fig. 3A, inset) were confirmed in the

Figure 1. T-type Ca2þ channelantagonists inhibit growth andinduce p53-dependent apoptosisin HCT116 cells. HCT116 cellswere treated with increasingmibefradil (Mib; A) or TTL1177 (C)concentrations. Drug-inducedgrowth inhibition was measuredafter 96 hours treatment usingsulforhodamine B (SRB) stainingassay. Plots present data from atleast three independentexperiments � SEM. To measureapoptosis, HCT116 cells weretreated with Mib (B) or TTL1177 (D)and after 24 hours, apoptosisinduction was assessed usingfluorescence caspase-3/7 activityassay as described in Materialsand Methods. Plots present datafrom at least three independentexperiments � SEM. Statisticalsignificance: �, P < 0.05;��, P < 0.001.






selected clones. Next, the parental CaCo2 and thoseexpressing p53wt were transiently transfected with twodifferent specific siRNA to knockdown CACNA1Gexpression. The extent of CACNA1G knockdown wasdemonstrated with RT-qPCR (Fig. 3B). Expression ofp53 sensitized CaCo2 cells to knockdown of CACNA1Ggene expression (Fig. 3C), with cell survival reduced for si(1) to 33% � 4.3% (CaCo2p53wt-11) versus 74% �6.1% for control cells and for si(2) cell survival at 64% �1.4% (CaCo2p53wt-11) versus 93%� 6.3% for controls.Importantly, the decrease in survival of CaCo2p53wt-11cells was associated with increased expression of p53-dependent genes, such as CDKN1A (p21Cip1/Waf1) andBBC3 (PUMA; Fig. 3D), indicating that p53 transcrip-tional activity was induced in response to knockdown ofCACNA1G.

Inhibition of T-type Ca2þ channels increases p53binding at the promoters and regulatory sequences oftarget genesIn response to mibefradil treatment, p53 protein accu-

mulated in HCT116 cells and its phosphorylation atSer392 increased in a mibefradil dose-dependent manner(Fig. 4A). In agreement with previous reports (8), mibe-fradil also induced expression of the CDK inhibitorCDKN1A (p21Cip1/Waf1), and the proapoptotic proteinBBC3 (PUMA; Fig. 4A). The expression of p53-depen-dent genes induced by mibefradil was confirmed at themRNA level (Fig. 4B). Total RNA from either DMSO-(0.1%) or mibefradil- (10 mmol/L) treated cells wasisolated and analyzed for expression of p53-responsivegenes using RT-qPCR. As a positive control of p53function, irradiated cells (6 Gy/6 hours after irradiation)

Figure 2. RNAi-mediated downregulation of a T-type Ca2þ channel subunit gene, CACNA1G, results in p53-dependent decrease in cell proliferation andapoptosis induction. A, RT-qPCR reaction assessing relative expression level of different T-type Ca2þ subunits, CACNA1G and CACNA1H, present inHCT116 cells. The plot represents data from three independent experiments � SEM. Inset represents Western blot analysis of CACNA1G and CACNA1Hproteins expressed in HCT116 cells. HCT116 were transiently transfected with scrambled (scrmbl, 25 nmol/L) or two different siRNA targeting a single T-typeCa2þ channel subunit CACNA1G [siCACNA1G (1) and siCACNA1G (2) 25 nmol/L]. After 96 hours total RNA was isolated and used for RT-qPCRreaction. B, RT-qPCR confirms specific downregulation of CACNA1G gene expression with si(1) and si(2). The plot represents data from at least twoindependent experiments � SEM. C, growth inhibition induced by siCACNA1G (1) or siCACNA1G (2) was determined by counting cells 96 hours aftertransfection using trypan blue exclusion assay. The plot represents data from four independent experiments � SEM. D, apoptosis assay in HCT116 cellstransfected with 25 nmol/L of siCACNA1G (1). Cells were transfected 48 hours prior measuring caspase-3/7 activity. The plot presents data from threeindependent experiments � SEM. Statistical significance: �, P < 0.05; ��, P < 0.01.






were used, and demonstrated similar levels of enrichmentof gene expression as cells treated with mibefradil (data notshown). Increased expression of p53-dependent genesCDKN1A (p21Cip1/Waf1) and BBC3 (PUMA) havebeen observed before (8), but we also uncovered addi-tional p53-induced genes that were elevated by inhibitionof T-type Ca2þ channels, such as growth arrest and DNAdamage–inducible gene GADD45A (Fig. 4B). As a neg-ative control of p53-dependent gene expression, we eval-uated mibefradil-treated HCT116 p53�/� cells (Fig. 4Aand B). Lack of p53-dependent gene expression inHCT116 p53�/� cells was consistent with their resistanceto mibefradil-induced apoptosis (Fig. 1B).To explore in more detail the action of p53 in mibefradil-

induced gene expression, cells treated with mibefradil (10mmol/L) for 9 hours were subjected to ChIP. The specificsequences within selected genes containing p53-bindingsites (p53BS), or in close proximity to p53BS, were detectedusing qPCR in immunoprecipitated p53-DNA complexes

from either nontreated control, ionizing radiation (6 Gy/6hours after irradiation), or mibefradil-treated cells. Bothionizing radiation and mibefradil increased association ofp53 at specific p53BS (expressed as fold of enrichment). Inagreement with our previous observation, mibefradil treat-ment increased p53 binding to CDKN1A, GADD45A, andBBC3 genes (Fig. 4C), corresponding well with increasedgene expression (Fig. 4A and B). Surprisingly, not all selectedproapoptotic p53 target genes (e.g., FAS, TP53AIP1, orBAX) showed an increase in p53 binding upon mibefradiltreatment (Fig. 4C and data not shown).

p38-MAPK is required for signaling during T-typeCa2þ channels inhibitionWe found that mibefradil treatment of HCT116 cells led

to an increase in Ser392 phosphorylation of p53 (Fig. 4A).Activated p38-MAPK associates with CK2 to enhancephosphorylation of p53 at Ser392 (31–33). Therefore, weinvestigated whether p38-MAPK was involved in the

Figure 3. Expression of p53wt gene sensitizes CaCo2 cells to downregulation of T-type Ca2þ channels. p53-mutant CaCo2 cells were stably transfected withp53wt gene (CaCo2p53wt-11) orwith pcDNA3.1 vector (CaCo2Vec). A, evaluation ofmRNAexpression levels of the T-typeCa2þ channels inCaCo2 cells. RT-qPCR reaction was performed using total RNA isolated from cells as described in Materials and Methods. The plot represents data from at least threeindependent experiments � SEM. In the inset, Western blot analysis of p53 protein level in CaCo2 cells. B, RT-qPCR reaction confirms RNAi-mediateddownregulation of CACNA1G gene expression in CaCo2 cells with specific siRNA [siCACNA1G (1) and siCACNA1G (2)]. The plot represents data fromat least two independent experiments � SEM. C, transient transfection of CaCo2 cells with siRNA targeting CACNA1G decreases cell survival of cellswith restored p53wt gene. CaCo2 cells were transfected with siCACNA1G (1) or siCACNA1G (2) and 24 hours later reseeded for colonies formation.The plot represents data from at least three independent experiments� SEM. D, RNAi-mediated downregulation of CACNA1G subunit increases expressionof p53-dependent genes in CaCo2p53wt-11 cells. Total RNA was used for RT-qPCR reaction using specific primers for p53-dependent gene expressionlisted in Supplementary Table S1. The plot represents averaged data obtained from transfections with si(1) and si(2) targeting CACNA1G geneexpression that was independently repeated � SEM. Statistical significance: �, P < 0.05; ��, P < 0.01.






response to T-type Ca2þ channel inhibition. HCT116 cellswere preincubated with either a pharmacologic inhibitor ofp38-MAPK, SB203580, or specific siRNA to knockdownthe expression of both MAPK14 and MAPK11 (p38-MAPKa/b), and then treated with mibefradil. Inhibitionor knockdown of p38-MAPK significantly relieved inhibi-tion ofHCT116 cell proliferation bymibefradil (Fig. 5A andB). These results provide evidence for the involvement ofp38-MAPK in the cellular response tomibefradil. In supportof these data, either SB203580 or knockdown of p38-MAPKprevented accumulation of p53 and phosphorylationof Ser392 in response to mibefradil (Fig. 5C for SB203580and Fig. 5D for si-p38-MAPKa/b). We observed higherlevels of p53 Ser15 phosphorylation inHCT116 cells treatedwith mibefradil (Fig. 5D); however, these increases were notstatistically significant (P¼ 0.671,N¼ 3).Our data indicatethat p38-MAPK is required for signaling to p53 in responseto inhibition of T-type Ca2þ channels.Finally, HCT116 cells were subjected to combined treat-

ment with SB203580 and mibefradil and assayed for expres-sion of CDKN1A (p21Cip1/Waf1) and BBC3 (PUMA).Combination treatment significantly reduced the expressionof CDKN1A (p21Cip1/Waf1) and BBC3 (PUMA) genescompared with treatment with mibefradil alone. The reduc-

tion was clearly evident at both the mRNA (Fig. 6A) andprotein level (Fig. 6B). Taken together, our results point top38-MAPK activation of p53-dependent transcription incells upon inhibition of T-type Ca2þ channels.

DiscussionT-type Ca2þ channels are expressed in epithelial cancer

cells, such as prostate, breast, and colon carcinoma (5,6, 37, 43), but not in the corresponding normal tissues,making them a potentially selective target for anticancertherapy. Pharmacologic inhibition of T-type Ca2þ channelswas previously shown to inhibit cell growth in severaldifferent cancer cell lines (4–6). Although p53 was impli-cated in the response (8), the signaling pathway(s) connect-ingT-typeCa2þ channels to proliferation and cell death havenot been defined.Results from this study show that both p38-MAPK and

p53 are required for immediate response (growth inhibitionand induction of apoptosis) following inhibition of T-typeCa2þ channels in cancer cells. The evidence suggests thatp38-MAPK promotes phosphorylation of Ser392 in the p53protein to increase p53 levels that in turn activates tran-scription of genes leading to cell-cycle arrest and caspase-mediated apoptosis (Fig. 7). This signaling pathway

Figure 4. T-typeCa2þ channel inhibitor,Mib, increases p53 phosphorylation onSer392, expression of p53-dependent genes andp53 binding at the promotersand regulatory sequences within genomic DNA. A, Western blot analysis of the whole-cell extracts isolated from HCT116 cells treated with increasingconcentrations of Mib. Blot is representative of at least three independent experiments. B, gene expression of p53-dependent genes: Fas, CDKN1A, BBC3,and GADD45A upon Mib treatment in HCT116 cells. RT-qPCR was performed using total RNA isolated from Mib-treated cells (10 mmol/L/9 h) andspecific primers used for gene expression as listed in Supplementary Table S1. Graph represents data from at least three independent experiments� SEM. C, association of p53 with its binding sites within a genome induced by Mib treatment as measured by ChIP assay. Cells were treated with Mib(10 mmol/L) for 9 hours prior isolation of protein–DNA cross-links. As a positive control, HCT116 cells were irradiated with 6 Gy and collected 6 hours afterirradiation. qPCR reaction was done in triplicates using primers listed in Supplementary Table S2. Data were normalized to the input and to thenondrug-treated nonirradiated HCT116 p53wt control. The plot represents data from at least two independent experiments � SEM.






presumably is kept in a relatively inactive state by the activityof T-type Ca2þ channels. The HCT116 and CaCo2 epi-thelial cancer cells studied here both express two subunits ofT-type Ca2þ channels, CACNA1G (Cav3.1) and CAC-NA1H (Cav3.2). Either pharmacologic inhibition of thechannels or RNAi-mediated knockdown of the Cav3.1subunit was sufficient for activation of the signaling path-way. Interestingly and in agreement with our other pub-lished results (4), knockdown of CACNA1H (Cav3.2) hadsignificantly smaller effects compared with the knockdownof CACNA1G, suggesting differences between the func-tional role of these two subunits of T-type Ca2þ channels(data not shown).On the basis of our results, we propose thatCACNA1G (Cav3.1) subunit of T-type Ca2þ channelspromotes survival of epithelial cancer cells by suppressinga proapoptotic signaling pathway (Fig. 7), whereas CAC-NA1H (Cav3.2) may be necessary to allow for cell-cycletransitions. Disruption of this signaling pathway at thechannel, or p38-MAPK or p53 prevents apoptosis inresponse to inhibition or knockdown of T-type Ca2þ

channels.This cell death pathway shows strong dependence on p53

function. Expression of p53 in CaCo2 cells converted themfrom resistant to sensitive to T-type Ca2þ channel down-

regulation (Fig 3C). As demonstrated by ChIP and geneexpression analyses, p53 binds to specific promoter/enhancersequences and thereby facilitates transcription of genes suchas CDKN1A and BBC3. The increase in p53 transcriptionalactivities correlates with its phosphorylation at Ser392.Pharmacologic inhibition or knockdown of MAPK14/11(p38-MAPKa/b) significantly decreased both Ser392 phos-phorylation and protein levels of p53, corresponding toreduced expression of CDKN1A and BBC3 genes.Phosphorylation of p53 at C-terminal residue Ser392 is

believed to be important for tetramerization, stability, andtranscriptional activities of p53 (44–46). Mutation ofSer392 to Ala, but not to phosphomimetic Asp, increasedchemoresistance of some cancer cells and increased colonyformation in soft agar, an indication of aggressive growth andgreater oncogenic properties of p53 (46). In agreement, p53Ser392 seems to be not phosphorylated inmutant p53 that isexpressed in breast cancer (47), and most likely the loss ofphosphorylation mark at this residue is responsible formaintenance of p53 oncogenic properties (reviewed in;ref. 47). Thus, phosphorylation of p53 at Ser392 may beimportant not only to stimulate p53-dependent transcrip-tion, but also could potentially decrease oncogenic proper-ties of mutant p53.

Figure 5. p38-MAPK regulatesantiproliferative action of p53 upontreatment of cells with Mib. A,HCT116 p53wt cells were treatedwith specific inhibitor of p38-MAPK(SB203580, 10 mmol/L) for 1 hourand subsequently with Mib(10 mmol/L) or (B) transfected withspecific siRNA to decreaseexpression of p38-MAPKa/b(10 nmol/L) and then treated withMib (10 mmol/L) for 24 hours. Nextday cells were trypsinized, countedusing trypan blue exclusion assayandnumber of live cells plotted as apercentage of control. Plotspresent data from at least threeindependent experiments � SEM.C, Western blot analysis of whole-cell lysates isolated from HCT116p53wt cells treated with Mib or incombination with SB203580. Theplot presents quantification of p53and P-Ser392 p53 signal relative toactin and nondrug-treated controlfrom at least two independentexperiments � SEM. D, Westernblot analysis of whole-cell lysatesisolated from HCT116 p53wt cellstransfected with sip38-MAPKa/band treated with Mib (10 mmol/L/24h). The plot representsquantification of p53 and P-Ser392p53 signal relative to actin andnondrug-treated control from atleast three independentexperiments � SEM. Statisticalsignificance: �, P < 0.05; ns,P > 0.05.






p38-MAPK is involved in cellular response to UV-induced DNA damage and DNA replication inhibition,leading to p53 phosphorylation at Ser15, 37, and 46(29), and has been suggested to indirectly (through activa-tion of CK2) phosphorylate p53 at Ser392 (31–33). p38-MAPK was also reported to phosphorylate p53 at Ser33during osmotic shock (30). However, based on the recentreport in the literature (48), we cannot exclude other kinasesfrom being involved in phosphorylation of p53 Ser392

in response to inhibition of T-type Ca2þ channels. Thesephosphorylation events activate p53 to execute growthinhibition and apoptosis programs. It has been shown thatmouse embryonic fibroblasts (MEFs) derived fromMAPK14 knockout mice (p38-MAPKa�/�) are more resis-tant to apoptosis induced by several stimuli (49), whichcorrelated well with decreased expression of p53-regulatedproapoptotic genes.Our data not only suggest involvement of p38-MAPK

in p53-dependent growth inhibition and apoptosis induc-tion, but also implicate that T-type Ca2þ channels fuelprosurvival processes in cells, especially cancer cells. Thisoccurs most likely through cross-talk with MAPK, giventhat the inhibition of T-type Ca2þ channels results inactivation of stress-activated p38-MAPK and at the sametime downregulation of ERK1/2 kinase (data not shown).These effects could be due to the changes in activity ofCa2þ-dependent protein phosphatases, such as calcineurinor PP2A (AB70C) that were previously reported to reg-ulate the MAPK pathway (50–52). A recent study fromour group suggests that mibefradil inhibits Akt andmTORC2 signaling (4), indicating additional cross-talkof T-type Ca2þ channels with intracellular prosurvivalsignaling pathways. It remains to be elucidated how p38-MAPK is activated in response to T-type Ca2þ channelinhibition or knockdown, and what Ca2þ-dependent

Figure 6. p38-MAPK activityincreases expression of p53-dependent genes upon Mibtreatment. A, normalized geneexpression of CDKN1A and BBC3in HCT116 p53wt cells treated withSB203580 (10 mmol/L), Mib(10mmol/L) alone or combination ofboth for 9 hours. Graph representsdata from two independentexperiments � SEM. B, proteinanalysis of whole-cell extractsisolated from HCT116 p53wt cellstreated with SB203580, Mib(10 mmol/L) or with combination ofboth for 9 hours. Membranes werestripped and reprobed with anti-BBC3 (PUMA) antibody. Westernblot analysis represents data fromtwo independent experiments.Statistical significance: �, P < 0.05.

Figure 7. The model of p53 activation upon T-type Ca2þ channelinhibition.






factors transduce signals to either upstream MAP3K5(ASK1) kinase (14) or directly to p38-MAPK.Our findings have significant implications for mibefradil-

based cancer therapy, since we observe activation of p38-MAPK, inhibition of cell growth and an increase in apoptosisupon treatment of cancer cells with the drug. The datasupport the notion for use of T-type Ca2þ channelsinhibitors as potentially effective agents to sensitize cellsto DNA damage in novel therapeutic approaches, as testedin interlaced therapy (9, 53). However, combinationtreatment of mibefradil with any inhibitor(s) of p38-MAPK that might be used to counteract proinflammatoryresponses in cancer (reviewed in ref. 54) should be carefullyconsidered.

Disclosure of Potential Conflicts of InterestJaroslaw Dziegielewski has received a commercial research grant from Tau

Therapeutics LLC. No potential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: B. Dziegielewska, J.M. Larner, J. DziegielewskiDevelopment of methodology: B. Dziegielewska, J.M. LarnerAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): B. DziegielewskaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): B. Dziegielewska, D.L. Brautigan, J.M. Larner, J. DziegielewskiWriting, review, and/or revision of the manuscript: B. Dziegielewska,D.L. Brautigan, J.M. Larner, J. DziegielewskiStudy supervision: J. Dziegielewski

AcknowledgmentsThe authors thank Tau Therapeutics LLC (Charlottesville, VA) for providing us

with mibefradil and TTL1177, Dr. Anindya Dutta (University of Virginia, Charlot-tesville, VA) for providing us with pcDNA3.1 p53wt plasmid, Dr. Amol Hosing fortechnical assistance, and Dr. Erin Griner for help with editing the article.

Grant supportThis work was supported in part by the UVA Department of Radiation Oncology

Dr. George Amornio pilot grant (to B. Dziegielewska) and UVA P30 CA44579 andpilot funding from the James and Rebecca Craig fund (to J. Dziegielewski).

Received September 11, 2013; revisedNovember 19, 2013; acceptedDecember 11,2013; published OnlineFirst December 20, 2013.

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2014;12:348-358. Published OnlineFirst December 20, 2013.Mol Cancer Res Barbara Dziegielewska, David L. Brautigan, James M. Larner, et al. Colon Cancer CellsGrowth Arrest and Apoptosis through Activation of p38-MAPK in

Channel Inhibition Induces p53-Dependent Cell2+T-Type Ca

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