Adenovirus-Mediated PTEN Treatment Combined with Caffeine Produces A

Adenovirus-mediated PTEN treatment combined with caffeine produces a synergistic therapeutic effect in colorectal cancer cells Yuji Saito,1 Began Gopalan,1 Abner M Mhashilkar,2 Jack A Roth,1 Sunil Chada,2 Louis Zumstein,2 and Rajagopal Ramesh1 1Department of Thoracic and Cardiovascular Surgery, The University of Texas M.D. Anderson Cancer Center, Houston, Texas, USA;and 2Introgen Therapeutics, Inc., Houston, Texas, USA.

The tumor suppressor phosphatase and tensin homologue deleted from chromosome 10 (PTEN) gene is a negative regulator of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt/PKB) signaling pathway. Overexpression of PTEN in cancer cells results in cell-cycle arrest and cell death through inhibition of PI3K. Caffeine, a xanthine analogue, is well known to enhance the cytocidal and growth-inhibitory effects of DNA-damaging agents such as radiation, UV light, and anticancer agents on tumor cells by abrogating DNA-damage checkpoints through inhibition of ataxia-telangiectasia-mutated (ATM), and ATM and Rad3-related (ATR) kinase activity. In this study, we demonstrate that treatment with a combination of adenovirus-mediated transfer of PTEN (Ad-PTEN) and caffeine synergistically suppressed cell growth and induced apoptosis in colorectal cancer cells but not in normal colorectal fibroblast cells. This synergistic effect was induced through abrogation of G2/M arrest, downregulation of the Akt pathway, and modulation of the p44/42MAPK pathway. Thus, combined treatment with Ad-PTEN and caffeine is a potential therapy for colorectal cancer. Cancer Gene Therapy (2003) 10, 803–813. doi:10.1038/sj.cgt.7700644 Keywords: PTEN; caffeine; colorectal cancer; apoptosis; synergy; gene therapy

he phosphatase and tensin homologue deleted from damaged DNA.13,14 The checkpoint may contribute to Tchromosome 10 (PTEN) gene is a tumor-suppressor the development of drug resistance, a formidable limita- gene located on human chromosome 10q23. 3.1 Frequent tion in current cancer treatment. If this is so, agents that deletions and somatic mutations of PTEN have been over-ride cell-cycle checkpoints could be used to sensitize reported in glioblastoma, endometrial cancer, prostate cells to killing by genotoxic drugs.15 Proof of this concept cancer, and small cell lung cancer.2–5 Overexpression of has arisen from studies with caffeine. Caffeine, a PTEN in cancer cells carrying mutant- or deletion-type methylxanthine, is known to have a broad range of PTEN inhibited cell proliferation and tumorigenicity via biochemical and physiological activities.16 Caffeine is well 6–9 induction of cell-cycle arrest at G1 and apoptosis. More documented to modulate carcinogenesis at various organ recently, studies using ovarian, thyroid, and colorectal sites, including liver, skin, lung, and mammary gland, in cancer cells that express wild-type PTEN (wt-PTEN) animals treated with carcinogens.16 Treatment with demonstrated that ectopic expression of PTEN resulted in caffeine significantly decreases lung tumor formation in growth inhibition and cell death.9–11 Furthermore, we mice treated with the precursors of N-nitrosomorpholine have recently demonstrated that ectopic expression of or with N-nitrosodiethylamine, 4-nitroquinoline-l-oxide, 17–20 PTEN induced G2 arrest and apoptosis in colorectal or urethane. Caffeine seems to invariably protect cancer cells that express wt-PTEN.12 against lung tumorigenesis in both mice and rats treated Most effective anticancer therapies are genotoxic agents with carcinogens. In contrast, caffeine can either stimulate that damage DNA and kill dividing cells rapidly. In or inhibit carcinogen-induced mammary gland tumori- addition to apoptosis, DNA damage induced by ionizing genesis, depending on the species and strains and the cell- radiation (IR) or other insults triggers, in addition to cycle phases during which it is administered.21 In recent apoptosis, cell-cycle checkpoint activation and subsequent years, caffeine has been shown to inhibit gastric tumor cell-cycle arrest, enhancing the ability of cells to repair promotion by sodium chloride in rats,22 despite enhancing the pancreatic tumorigenesis caused by N-nitrosobis (2- oxopropyl) amine in hamsters when administered during Received April 3, 2003. the postinitiation phase.23 Caffeine also protects against Address correspondence and reprint requests to: Dr. Rajagopal 24,25 Ramesh, PhD, Department of Thoracic and Cardiovascular Surgery, UV light-induced skin tumorigenesis. More recent The University of Texas M.D. Anderson Cancer Center, 1515 studies have demonstrated that caffeine is an inhibitor Holcombe Blvd, Box 445, Houston, TX 77030, USA. of cell-cycle checkpoints, causing disruption of DNA E-mail: [email protected] damage at checkpoints (including the G2/M checkpoint) PTEN and caffeine synergism in colorectal cancer cells Y Saito et al 804 through inhibition of ataxia-telangiectasia-mutated 3 hours of infection with Ad-PTEN or Ad-Luc at various (ATM), and ATM and Rad3-related (ATR) kinase multiplicity of infection (MOI) units (i.e., viral particles activity and sensitizes tumor cells to IR, cisplatin, and [vp]/cell), 50-ml aliquots of medium containing varying other genotoxic agents.26–29 Although the tumor-suppres- concentrations of caffeine were added into each well. Cells sive effect of PTEN and the sensitization to various were then incubated at 371C in a humidified atmosphere genotoxic agents by caffeine have been studied in a wide containing 5% CO2. At 3 days after incubation, cell variety of cancer cells, caffeine enhancement of PTEN- growth and viability were quantified by XTT assay. induced apoptosis has not been previously studied. Briefly, the culture medium was removed, and 150 mlof Therefore, we tested whether treatment with a combina- XTT reaction mixture was added into each well with fresh tion of adenovirus-mediated transfer of PTEN (Ad- medium at a final concentration of 0.3 mg/ml/well. Cells PTEN) and caffeine would enhance the therapeutic effect. were then incubated for 2 hours at 371C. The absorbance In the present study, we demonstrate that treatment with was measured at a wavelength of 450 nm against a combinations of Ad-PTEN and caffeine induces synergis- reference wavelength of 630 nm in a microplate reader tic suppression of cell growth and apoptosis selectively in (Model ELX808; Bio-Tek Instruments, Inc., Winooski, colorectal cancer cells, but not in normal cells, through VT). Percentage cell viability was calculated in terms of abrogation of G2/M arrest, downregulation of the Akt the absorbancy in treated cells relative to the absorbancy pathway, and modulation of the p44/42MAPK pathway. in untreated control cells. Experiments were repeated at These results suggest that treatment with a combination least three times for each treatment in each individual of Ad-PTEN and caffeine can be an effective gene experiment. therapeutic strategy for human colorectal cancer. Isobologram analysis The combination effects were analyzed by a modified Materials and methods isobologram method.32 Briefly, three isoeffect curves Cell lines and cell culture (modes I, IIA, and IIB), which were derived from each growth-inhibition curve, were drawn; the total area Colorectal cancer cell lines SW480 and DLD-1, normal enclosed by these three lines represented an ‘‘envelope colon fibroblast cell line CCD-18Co, and prostate cancer of additivity.’’ Actual IC values were obtained and cell lines DU145 and LNCaP were obtained from the 50 plotted on the envelope. If the experimentally observed American Type Culture Collection (Rockville, MD). IC was plotted on the left side of the envelope, the Colon cancer cell lines HCT116 Á p53 ðþ=þÞ and 50 combination was considered to show a supra-additive HCT116 Á p53 ðÀ=ÀÞ were gifts from Dr. Bert Vogelstein (synergistic) interaction. If it was plotted within the (Johns Hopkins Medical Oncology Center, Baltimore, envelope, the combination was regarded as additive, and MD). All cell lines were grown in RPMI 1640 medium, if it was plotted on the right of the envelope and within except SW480 and CCD-18Co, which were maintained in the dotted-line square, the combination was considered to Dulbecco’s modified Eagle’s medium and in modified be subadditive. If the observed IC was plotted outside essential medium in Earle’s balanced salt solution with 50 the square, the combination was considered to be nonessential amino acids, respectively. The growth protective. medium was supplemented with 10% fetal bovine serum, antibiotics, and l-glutamine (Gibco -BRL, New York, NY). Gene transduction Preliminary experiments using an adenoviral vector Construction of recombinant adenoviral vector carrying the green fluorescent protein (Ad-GFP) showed Construction and production of the recombinant adeno- that, at an MOI of 5000 vp/cell, the adenovirus can infect viral vectors carrying PTEN (Ad-PTEN) or the luciferase 99.3% of HCT116 p53 ðþ=þÞ and 81.9% of CCD-18Co gene (Ad-Luc) have been described elsewhere.30 cells and more than 80% of other cells (data not shown) by 24 hours after infection. On the basis of these results, XTT assay we used Ad-PTEN or Ad-Luc at an MOI of 5000 vp/cell in all subsequent experiments. Inhibition of tumor cell growth by treatment with combinations of Ad-PTEN, Ad-Luc, or IR and caffeine was analyzed by quantitatively determining cell viability Cell-cycle analysis using an improved XTT assay (Roche Molecular Bio- Cells were seeded in 10-cm culture dishes (5 Â 105– chemicals, Indianapolis, IN).31 Cells were plated in 96- 10 Â 105 cells per dish) and treated with 2 mM caffeine well microtiter plates at densities of 2 Â 103–6 Â 103 cells 3 hours after infection with Ad-PTEN or Ad-Luc,or per well in 100 ml of medium. At 24 h after the cells were treated with 20 mM of LY294002 (PI3K inhibitor) or plated, 50-ml aliquots of medium containing varying 10 mM U0126 (MEK1/2 inhibitor) (Cell Signaling Tech- concentrations of Ad-PTEN or Ad-Luc were added to nology, Beverly, MA). To produce DNA damage, cells each well, or cells were exposed to varying doses of IR were exposed to 2.0 Gy IR 30 minutes after treatment with after 30 minutes of exposure to 100-ml aliquots of medium caffeine. At specified times after treatment, cells were containing varying concentrations of caffeine. After harvested by trypsinization, washed once with ice-cold

Cancer Gene Therapy PTEN and caffeine synergism in colorectal cancer cells Y Saito et al 805 phosphate-buffered saline (PBS), fixed with 70% ethanol, Mode 1 Mode 2a and stored at À201C. Cells were then washed twice with Mode 2b data ice-cold PBS and treated with RNase (30 minutes at 371C, 500 U/ml) (Sigma Chemicals, St Louis, MO), and DNA was stained with 50 mg/ml propidium iodide (Boehringer- a HCT116(+/+) HCT116(−/−) Mannheim, Indianapolis, IN). DNA contents and cell- cycle phases were analyzed on a fluorescence-activated 1 1 cell sorter (FACScan, EPICS XL-MCL; Beckman Coul- ter, Fullerton, CA). 0.5 0.5 Apoptotic staining Cells were seeded in six-well tissue culture dishes at a 5 density of 1 Â 10 cells per well and treated with 2 mM 0 0 caffeine or PBS as a mock control 3 hours after infection 0 0.5 1.0 1.5 0 0.5 1.0 1.5 of Ad-PTEN. At 72 hours after infection, cells were analyzed for apoptosis using Hoechst 33258 staining SW480 DLD-1 (Sigma Chemicals). Apoptotic cells were identified via apoptotic body and/or chromosome condensation. 1 1

Western blot analysis 0.5 Cells were plated at densities of 5 Â 105–10 Â 105 in 10-cm 0.5 dishes overnight. They were treated with 20 mM LY294002 or 10 mM U0126 alone, with 2 mM caffeine 3 hours after infection with Ad-PTEN or Ad-Luc,or 0 0 exposed to 2.0 Gy IR at 30 minutes after treatment with 0 0.5 1.0 1.5 0 0.5 1.0 1.5 caffeine. Cells were incubated for the indicated times at CCD-18Co HCT116(+/+) 371C and then collected, and whole-cell lysates prepared. b c The cell lysates were analyzed for the expression of 1 various proteins by Western blot analysis. The blots were 1 reprobed using antibodies against b-actin (Sigma Chemi- cals) to ensure equal loading and transfer of proteins. The following primary antibodies were purchased and used at 0.5 0.5 1:500 or 1:1000 dilution: PTEN, Cdc2, p53 (Bp53-12), and p27Kip1, (Santa Cruz Biotechnology, Santa Cruz, CA); Cdc25C, phospho-Akt, and phospho-p44/42MAPK (Cell 0 1.0 0 Signaling Technology); cyclin B1 (Neo Markers, Fre- 0 0.5 1.5 0 0.5 1.0 1.5 WAF1 mont, CA); and p21 (Oncogene Research Products, Figure 1 Isobologram analysis of the effects of treatment with two- Boston, MA). agent combinations on colorectal cell lines. Cells seeded in 96-well plates were treated with various concentrations of Ad-PTEN, caffeine, Ad-PTEN, or Ad-Luc and caffeine. At 72 hours after

treatment, isobolograms at IC50 levels were generated in colorectal Results cancer cell lines (HCT116 Á p53 ðþ=þÞ, HCT116 Á p53 ðÀ=ÀÞ, Combined treatment with Ad-PTEN and caffeine SW480, DLD-1) (a) and (c), and normal colorectal fibroblast cells produces a synergistic effect in colorectal cancer cells (CCD-18Co), (b), treated with combinations of Ad-PTEN and caffeine (a) and (b), or Ad-Luc and caffeine (c). To observe the effect of the combination of Ad-PTEN and caffeine, four colorectal cancer cell lines (HCT116 p53 ðþ=þÞ, HCT116 p53 ðÀ=ÀÞ, SW480, DLD-1) and one normal colon cell line (CCD-18Co) were analyzed cells under these treatment conditions (Fig 1b). An 72 hours after treatment (Fig 1a, b). All of the experi- additive effect was also observed in LNCaP cells treated ments were repeated at least three times for each with Ad-PTEN and caffeine (data not shown). To further cell line. At the IC50 concentration for each cell line, confirm that the observed synergistic effect was specific, combination of Ad-PTEN and caffeine produced a we tested the effects of a combination of Ad-Luc and complete synergistic effect in HCT116 p53 ðþ=þÞ and caffeine in HCT116 p53 ðþ=þÞ cells (Fig 1c). Unlike SW480 cells but a boundary effect between addition Ad-PTEN plus caffeine, Ad-Luc plus caffeine produced and synergy in HCT116 p53 ðÀ=ÀÞ and DU145 cells. The a boundary effect between additive and protective observed data points were distributed either within effects. These results indicate that treatment with a the additive envelope or scattered around the boundary combination of Ad-PTEN and caffeine specifically between the additive and the synergistic areas (Fig 1a). induces a synergistic effect in tumor cells but not in However, a protective effect was observed in CCD-18Co normal cells (Table 1).

Cancer Gene Therapy PTEN and caffeine synergism in colorectal cancer cells Y Saito et al 806 Table 1 Therapeutic effect produced in various cell lines when treated with a combination of Ad-PTEN and caffeine is independent of PTEN and p53 status

Cell lines PTEN alleles p53 status Cell cycle arrest Isobologram

HCT116(+/+) Wild Wild G2 Synergistic HCT116(À/À) Wild Deletion G2 Synergistic and additive SW480 Wild Mutant G2 Synergistic DLD-1 Wild Mutant G2 Synergistic and additive a CCD-18Co Wild Wild G2 Protective b DU145 Wild Mutant G2 Synergistic and additive

b LnCAP Mutant Wild G1 Additive

aNormal colon fibroblast cell line. bProstate cancer cell not sequenced for PTEN.

Treatment with a combination of Ad-PTEN and caffeine inhibitor of phosphatidyl inositol-3-kinase or PI3K (data induces apoptosis in colorectal cancer cells not shown). Although one of the PTEN functions is PI3K inhibition, Ad-PTEN, unlike LY294002, did not induce To determine whether treatment with Ad-PTEN and G arrest in HCT116 p53 ðþ=þÞ and CCD-18Co cells caffeine induced apoptosis, HCT116 p53 ðþ=þÞ and 1 carrying wt-PTEN.9,11,33 These results, which suggest that CCD-18Co cells were analyzed 72 hours after treatment Ad-PTEN induces G /M arrest in cells that express wt- for apoptotic changes by using fluorescence-activated cell 2 PTEN, are in agreement with the results of our recent sorting (FACS) and Hoechst 33258 staining. A significant studies.12,30 In contrast, treatment with a combination of increase (P .01) in the number of cells in sub-G /G o 0 1 Ad-PTEN or Ad-Luc and caffeine yielded dramatically phase, an indicator of apoptotic changes, was observed by lower numbers of HCT116 p53 ðþ=þÞ and CCD18-Co FACS analysis in HCT116 p53 ðþ=þÞ cells treated with cells that underwent G /M-phase arrest than treatment Ad-PTEN alone (7.96%) or with a combination of Ad- 2 with Ad-PTEN or Ad-Luc alone. Treatment with caffeine PTEN and caffeine (13.05%) (Fig 2a). Cells treated with alone yielded no significant decrease in the number of caffeine, Ad-Luc, or a combination of Ad-Luc and cells in the G /M phase when compared to control cells caffeine demonstrated no significantly higher number of 2 treated with PBS. However, the number of cells in G /M apoptotic cells than PBS-treated control cells. In CCD- 2 phase was significantly higher (P .01) in cells treated 18Co cells, treatment with Ad-PTEN or Ad-PTEN and o with Ad-PTEN and caffeine than in cells treated with Ad- caffeine yielded no significantly higher number of Luc and caffeine. apoptotic cells than control cells (Fig 2a). No increase in apoptotic cells was observed in CCD-18Co, even on day 5 after combined treatment, suggesting that normal Signaling pathways regulated by PTEN overexpression cells are resistant to such treatments (data not shown). To and caffeine treatment in colorectal cancer cells and confirm these results, cells were stained with Hoechst normal cells 33258. Tumor cells (HCT116 p53 [+/+]) but not normal To investigate the mechanism through which PTEN- cells (CCD-18Co) revealed condensed and fragmented induced G /M arrest is abrogated by caffeine, proteins nuclei, an indicator of cells undergoing apoptosis, when 2 related to the G /M and G cell-cycle checkpoints were treated with Ad-PTEN, or with Ad-PTEN and caffeine 2 1 evaluated. In this analysis, HCT116 p53 = and (Fig 2b). Cells treated with PBS or caffeine showed no ðþ þÞ CCD18-Co cells were treated with 2 mM caffeine, PBS, apoptotic morphology. Ad-PTEN or Ad-Luc alone, or a combination of Ad- PTEN or Ad-Luc and caffeine. Total cell lysates were Caffeine abrogates Ad-PTEN-induced G2/M cell-cycle prepared 48 hours after treatment and analyzed by arrest Western blot analysis. Cells treated with Ad-PTEN,or We next determined the effects of Ad-PTEN and caffeine Ad-PTEN and caffeine, demonstrated exogenous PTEN treatment on cell-cycle phases in HCT116 p53 ðþ=þÞ and protein expression that resulted in inhibition of pAKT in CCD-18Co cells by using FACS analysis. Cell-cycle both cell types (Fig 4a). However, the PTEN protein analysis demonstrated a significantly greater G2/M expression was significantly increased in HCT116 p53 population (Po.05) in 28.9% of HCT116 p53 ðþ=þÞ ðþ=þÞ cells, but not in CCD18-Co cells, when treated cells and in 17.85% of CCD18-Co cells 72 hours after with the combination of Ad-PTEN and caffeine. Treat- treatment with Ad-PTEN alone (Fig 3a, b) than in control ment with Ad-PTEN alone increased expression of p53, cells treated with Ad-Luc, caffeine, PBS, or a combination p21 and p27 and decreased expression of both phos- of Ad-PTEN or Ad-Luc and caffeine. However, HCT116 phorylated and nonphosphorylated Cdc25C when com- p53 ðþ=þÞ and CCD-18Co cells treated with LY294002 pared to cells treated with PBS or Ad-Luc. However, no were arrested at G1 phase, since LY294002 is an PI3K change in the expression of phospho-Chk1 and phospho-

Cancer Gene Therapy PTEN and caffeine synergism in colorectal cancer cells Y Saito et al 807 increased levels of both phosphorylated and nonpho- a 14 PBS sphorylated Cdc25C. In contrast, CCD18-Co cells treated 12 Ad-Luc with caffeine demonstrated decreased expression of p21, Ad-PTEN Cdc25C, and cyclin B1 with no change in p53. Cells 10 Caffeine treated with combination of Ad-PTEN and caffeine 8 Ad-Luc + Caffeine demonstrated higher expression of p53 and p27 than cells Ad-PTEN + Caffeine treated with PBS. Increased expression of p53 and p27 6 was due to PTEN since the expression of these proteins 4 did not increase in cells treated with PBS alone. However,

% Apoptotic Cell Number expression of p21, both phosphorylated and nonpho- 2 sphorylatedCdc25C, cyclin B1, and Cdc2 decreased after 0 treatment with a combination of Ad-PTEN and caffeine. CCD-18Co HCT116(+/+) Treatment with LY294002 decreased expression of cyclin B1, but did not change the expression level of Cdc25C, CCD-18Co HCT116(+/+) PBS 0.16 2.36 when compared with control cells (data not shown). Ad-Luc 0.145 3.055 To further determine the underlying mechanism Ad-PTEN 0.19 7.965 through which treatment with a combination of Ad- Caffeine 0.23 2.77 Ad-Luc + Caffeine 0.25 2.555 PTEN and caffeine produced a synergistic effect, we Ad-PTEN + Caffeine 0.425 13.05 examined the phosphorylation status of p44/42MAPK and FAK, which PTEN inhibits mainly through depho- 34,35 b HCT116(+/+) CCD-18Co sphorylation. Although phospho-p44/42MAPK expression was not changed by Ad-PTEN treatment alone, it was significantly increased in HCT116 p53 ðþ=þÞ but not in by treatment with the combination of Ad-PTEN PBS and caffeine (Fig 4b). No significant change in phospho- p44/42MAPK was observed in CCD18-Co cells when treated with Ad-PTEN and caffeine. Therefore, to determine whether the synergistic effect of Ad-PTEN and caffeine in HCT116 p53 ðþ=þÞ was due to increased phosphorylation of p44/42MAPK, we used FACS analysis to investigate phosphorylation status of p44/42MAPK and apoptotic ratio by treating cells with 10 mM U0126, Caffeine which is a MEK1/2 inhibitor. Treatment with Ad-PTEN, caffeine, and U0126 significantly increased the phosphorylation status of p44/42MAPK; however, expression of p44/42MAPK was less than in cells treated with Ad- PTEN and caffeine only (Fig 4b). In contrast, FACS analysis showed a significantly greater apoptotic ratio Ad-PTEN + (39.35%) (Po.01) in cells treated with a combination of Caffeine Ad-PTEN, caffeine, and U0126 than in cells treated with Ad-PTEN and caffeine only (13.05%; Fig 4c). No significant change in the expression of phospho-FAK was observed among the treatment groups 48 hours after treatment (data not shown), although phospho-FAK was Figure 2 Induction of apoptosis by treatment with combinations of decreased at later time points in HCT116 p53 ðþ=þÞ cells Ad-PTEN and caffeine. (a) The numbers of cells at phase sub-G0/G1 treated with caffeine alone or with Ad-PTEN and caffeine (apoptotic cells) in colorectal cancer cells HCT116 Á p53 ðþ=þÞ and (data not shown). normal colorectal fibroblast cells CCD-18Co were analyzed by flow cytometry 72 hours after treatment with PBS, Ad-Luc, Ad-PTEN, caffeine, or combinations of caffeine with Ad-Luc or Ad-PTEN. Data Comparison of the combined effects of Ad-PTEN and represent the means of two experiments. Error bars denote standard caffeine treatment and the combined effects of radiation error (SE). (b) Apoptotic analysis of HCT116 Á p53 ðþ=þÞ cancer cells and CCD-18Co normal cells by Hoechst 33258 staining was and caffeine in colorectal cancer cells performed 72 hours after treatment with PBS, caffeine, or combina- To test whether caffeine can abrogate G2/M phase arrest tions of Ad-PTEN and caffeine. Treatment with combinations of Ad- induced by other agents such as IR, we evaluated the PTEN and caffeine induced apoptosis in cancer cells but not in combined effects of radiation treatment and caffeine and normal cells. Arrows indicate apoptotic cells (magnification Â 400). compared those effects to the effects of PTEN and caffeine in HCT116 p53 ðþ=þÞ cells. Cells were treated Chk2 was observed (data not shown). HCT116 p53 with Ad-PTEN (5000 vp/cell) and caffeine or IR (2 Gy) ðþ=þÞ cells treated with caffeine alone demonstrated and caffeine and subjected to cell-cycle analysis 48 hours decreased expression of p53, p21, and cyclin B1 and after treatment. Cells treated with Ad-PTEN (51.4%) or

Cancer Gene Therapy PTEN and caffeine synergism in colorectal cancer cells Y Saito et al 808 HCT116(+/+) with concomitant increases in the number of cells in G1 phase (Fig 5a, b). Note, although a shift in the number of PBS a cells from G2/M phase to G1 phase was observed when 80 Ad-Luc treated with Ad-PTEN or IR plus caffeine, there was no Ad-PTEN 70 significant increase in the number of cells in the sub-G1 Caffeine 60 phase. This is probably due to the early time point tested Ad-Luc + Caffeine since an increase in the number of apoptotic cells (sub-G ) 50 1 Ad-PTEN + Caffeine was observed at later time points (472 hours; data not 40 shown). These results indicate that caffeine abrogates G2/ 30 M arrest induced by various therapeutic agents. (%) Cell Number 20 To further investigate whether treatment with the 10 combination of IR and caffeine produced a synergistic 0 effect similar to that observed with Ad-PTEN and G1 S G2/M caffeine, HCT116 p53 ðþ=þÞ cells were treated with PBS, Ad-PTEN, IR, or a combination of Ad-PTEN or IR G1 S G2/M and caffeine. At 72 hours after treatment, cells were PBS 60.15 26.85 13 analyzed by XTT assay for synergism using isobologram Ad-Luc 58.8 27 14.2 analysis and by FACS to determine the apoptotic ratio. Ad-PTEN 52.1 19 28.9 Caffeine 59.67 26.33 14 Combination treatment with IR and caffeine produced an Ad-Luc + Caffeine 63.1 27.9 9 additive effect (Fig 6a). FACS analysis 72 hours after Ad-PTEN + Caffeine 65.7 20.2 14.1 treatment revealed no significant difference in the apoptotic ratio in cells treated with a combination of PTEN b CCD-18Co Ad- and caffeine and in cells treated with a combination of IR and caffeine (Fig 6b). After 120 hours, 100 however, the apoptotic ratio was significantly higher in 90 cells treated with a combination of Ad-PTEN and 80 70 caffeine (99%), than in cells treated with IR and caffeine 60 (15.45%). Furthermore, we observed induction of phos- 50 phorylated p44/42MAPK expression when cells were 40 subjected to IR treatment alone or to a combination of

(%) Cell Number 30 IR and caffeine (Fig 6c). Induction of p44/42MAPK was 20 higher in cells treated with IR and caffeine and was 10 similar to that seen when cells were treated with Ad- 0 PTEN and caffeine. G1 S G2/M

G1 S G2/M Discussion PBS 78.3 12.5 9.2 Ad-Luc 81.2 10.9 7.9 Ad-PTEN 58.6 23.55 17.85 In the present study, we investigated the effects of Caffeine 92.4 3.55 4.05 treatment with a combination of Ad-PTEN and caffeine Ad-Luc + Caffeine 84.85 11.05 4.1 in colorectal cancer cells that express wt-PTEN. Treat- Ad-PTEN + Caffeine 92.9 3.15 4.05 ment with this combination successfully produced complete synergistic suppression and induced apoptosis to a Figure 3 Induction and abrogation of G2/M cell-cycle arrest due to overexpression of PTEN and treatment with caffeine. (a) significantly higher extent in colorectal cancer cells HCT116 Á p53 ðþ=þÞ colorectal cancer cells and (b) CCD-18Co (HCT116 p53 ðþ=þÞ) than in normal colorectal cells normal colorectal fibroblast cells were treated with PBS, Ad-Luc, Ad- (CCD-18Co). To further compare the effects of this PTEN, caffeine, combinations of caffeine with Ad-Luc or Ad-PTEN, combination in cells that express wild-type p53 (wt-p53) or 20 mM LY294002. Cells were harvested 72 hours after treatment and cells that express mutant p53 (mt-p53), we used and cell-cycle analysis was performed by using flow cytometry. In HCT116 p53 ðþ=þÞ and HCT116 p53ðÀ=ÀÞ cells. To total 20,000 events were captured for each treatment, and the data compare the effects of this combination in wt-PTEN cells are shown as histograms. The cell-cycle phase is represented on the and in mutant-type PTEN cells, we used prostate cancer X-axis. Data were generated in duplicate; the average values are cell lines LNCaP, expressing mutated PTEN, and DU145, shown. Bars denote standard error (SE). expressing wt-PTEN. We showed not only that overexpression of PTEN significantly suppressed growth and induced apoptosis in tumor cells compared to normal IR (33.4%) alone were arrested at the G2/M phase (Fig cells, but also that treatment with a combination of 5a, b). When these treatments were combined with overexpression of PTEN and caffeine produces a syner- caffeine, however, the numbers of cells in the G2/M gistic inhibitory effect in cancer cells expressing wt-PTEN phase in the Ad-PTEN-treated (27%) and IR-treated (Table 1). Although in the present study we have tested (23.6%) groups were significantly decreased (Po.01), two prostate cell lines that differ in their PTEN status to

Cancer Gene Therapy PTEN and caffeine synergism in colorectal cancer cells Y Saito et al 809

a e

+ Caffeine + Caffeine + Caffeine + Caffein PTEN Luc PTEN Luc PTEN Luc PTEN Luc Ad- Ad- Caffeine Ad- Ad- PBS Ad- Ad- Caffeine Ad- Ad- PBS PTEN PTEN

p-Akt p-Akt

p53 p53

p21 p21

p27 p27

p-Cdc25c p-Cdc25c Cdc25c Cdc25c

Cyclin B1 Cyclin B1

Cd c2 Cd c2

β-actin β-actin

HCT116 CCD18-Co

b e

+ Caffeine + Caffeine + Caffeine + Caffein + Caffeine PTEN Luc PTEN Luc PTEN Luc PTEN Luc PTEN + U0126 Ad- PBS Ad- Ad- Caffeine Ad- Ad- PBS Ad- Ad- CaffeineAd- Ad- + U0126 P-p44/42 MAP K P-p44/42 MAP K

β-actin β-actin 1.0 0.79 Dens. Ratio HCT116 CCD18-Co c 45 Sub G0\G1 PBS 40 PBS 2.36 Ad-Luc 3.095 Ad-Luc 35 Ad-PTEN 7.985 Caffeine 2.77 Ad-PTEN 30 Ad-Luc + Caffeine 2.595 Ad-PTEN + Caffeine 13.05 25 PTEN + Caffeine + U0 39.35 Caffeine

20 Ad-Luc + Caffeine 15 Ad-PTEN + Caffeine (%) Apoptotic ratio 10 PTEN + Caffeine + U0126

5 U0126 0 Sub G0\G1

Figure 4 Signaling pathways regulated by PTEN overexpression and caffeine. HCT116 Á p53 ðþ=þÞ colorectal cancer cells and CC18-Co normal cells were treated with PBS, Ad-Luc, Ad-PTEN, caffeine, combinations of caffeine with Ad-Luc or Ad-PTEN,or10mM U0126. At 48 hours after treatment, cells were harvested and examined by Western blot analysis. (a) G1 and G2/M phase-associated proteins. (b) Phosphorylation status of p44/42MAPK. The corresponding b-actin levels are shown as a loading control. (c) The numbers of cells at sub-G0/G1 (apoptotic cells) in HCT116 Á p53 ðþ=þÞ colorectal cancer cells were analyzed by flow cytometry 72 hours after treatment with PBS, Ad-Luc, Ad-PTEN, caffeine, or combinations of caffeine with Ad-Luc, Ad-PTEN, or Ad-PTEN and10 mM U0126. Data represent the means of duplicate experiments. Error bars denote standard error (SE). support our findings on the ability of Ad-PTEN plus previous studies are supported by the observation that caffeine treatment to induce a synergistic effect in p53 plays an important role in DNA damage-induced colorectal cancer cells that are wt-PTEN, a note of cell-cycle checkpoints and contributes to the prevention caution is that these cells may have additional differences of polyploidy formation.30–41 More recent studies have apart for their differences in the PTEN status. Further- demonstrated that PTEN protects p53 from Mdm2, more, this synergistic inhibitory effect was independent allows cells to respond to damage or mutation with of endogenous p53 status. This contrasts with previous an apoptotic response, and sensitizes cancer cells to reports demonstrating that p53-null or -mutant cells chemotherapy.41,43 In fact, Tsuchiya et al43 showed were more sensitive to caffeine-induced radiosensitization that the synergistic antitumor effect of caffeine and than wt-p53 cells.36–38 It is possible that the results of cisplatin was enhanced by reintroduction of wild-type

Cancer Gene Therapy PTEN and caffeine synergism in colorectal cancer cells Y Saito et al 810

Figure 5 Abrogation of G2/M cell-cycle arrest due to overexpression of PTEN or ionized radiation combined with caffeine. HCT116 Á p53 ðþ=þÞ colorectal cancer cells were (a) treated with Ad-PTEN or combination of Ad-PTEN and caffeine, or (b) exposed to 2 Gy IR or a combination of IR and caffeine. Cells were harvested 24 hours after treatment and cell-cycle analysis was performed by using flow cytometry. In total, 20,000 events were captured for each treatment, and the data are shown as histograms. The cell-cycle phase is represented on the X-axis. Data were generated in duplicate; the average values are shown. Bars denote standard error (SE).

p53 into human osteosarcoma cells. Thus, the role of p53 both threonine-14 and tyrosine-15 and then activates remains controversial. Cdc2/cyclin B1 complexes, leading to progression of the The mechanism by which the synergistic inhibitory cells to M phase. In response to DNA damage or effect was produced was determined by cell-cycle analysis. inhibition of DNA replication, however, inhibitory Treatment with Ad-PTEN alone resulted in G2/M-phase phosphorylation of cdc2 remains, and the cells are 44–47 arrest of both tumor cells and normal cells, while arrested at G2 phase. Our data demonstrate that treatment with caffeine alone resulted in G1 arrest. Cdc25C expression was downregulated following Ad- Induction of G2/M arrest by Ad-PTEN in cells that PTEN treatment in both tumor cells and normal cells. express wt-PTEN is not surprising and is in agreement ATM and ATR are proximal components of DNA with results of our recent studies.12,30 However, PTEN damage-induced cell-cycle checkpoint pathways, and induction of G2/M phase was abrogated by caffeine in activated ATM and ATR phosphorylate Chk2 and both tumor and normal cells. Caffeine-induced G1 was Chk1, respectively. Therefore, DNA damage leads to significantly more common in normal CCD-18Co cells activation of two related protein kinases, Chkl and Chk2, than in HCT116 p53 ðþ=þÞ cells, suggesting that caffeine which then phosphorylate the Cdc25C phosphatase on 42 had a stronger inhibitory effect on proliferation of CCD- serine-216. p53 is also required for G2 arrest in response 18Co cells proliferation than on that of HCT116 p53 to DNA damage;48 therefore, we assayed 14-3-3s, ðþ=þÞ cells. Interestingly, the increased inhibitory effect GADD45, and p21WAF1, which reside downstream of of caffeine in CCD-18Co cells did not result in any p53 and are associated with modulation of Cdc2 49 increase in the number of apoptotic cells, even on day 5 phosphorylation and G2/M-phase arrest. Ad-PTEN after treatment (data not shown). increased p53 and 14-3-3s expression, but not GADD45 WAF1 To further understand the mechanism by which the G2/ (data not shown) or p21 , in HCT116 p53 ðþ=þÞ M checkpoint can be altered by Ad-PTEN, we analyzed colorectal cancer cells compared with controls. We next the level of Cdc25C protein expression. In G2/M phase, evaluated the effect of caffeine, an inhibitor of ATM and active Cdc25C phosphatase dephosphorylates Cdc2 on ATR that abrogates the G2 checkpoint as described, on

Cancer Gene Therapy PTEN and caffeine synergism in colorectal cancer cells Y Saito et al 811 a G2 arrest induced by PTEN. Caffeine dramatically abolished G2 arrest in HCT116 Á p53 ðþ=þÞ cells and 1 modulated G2 checkpoint-associated proteins (p53, p21WAF1, p27 Kip1, both phosphorylated and nonphosphorylated Cdc25C, cyclin B1, and Cdc2), but we were not able to detect dephosphorylation of Chkl and Chk2 induced by caffeine. These results suggest that Ad-PTEN 0.5 induced G2/M arrest in cancer cells carrying wt-PTEN occurs by inhibition of Cdc25C and support the findings of our recent studies.12,33 Thus, the main targets of PTEN in cells carrying wt -PTEN may be different from those in cells carrying mutant- or deletion-type PTEN. We suggest that, on the basis of the status of endogenous PTEN, cells 0 0 0.5 1 1.5 could be arrested at either G1 or G2 phase. Similarly, the downstream pathways may be different. However, the Mode1 Mode2a exact mechanism by which PTEN causes this difference in Mode2b data cell-cycle arrest is unclear and remains to be investigated. Further analysis of the underlying mechanism of the observed synergistic inhibitory effect of Ad-PTEN and b caffeine treatment in tumor cells demonstrated inhibition 100 PBS of the Akt pathway and upregulation of the p44/ 90 Ad-PTEN 42MAPK pathway. However, inhibition of Akt was 80 Ad-PTEN + Caffeine 70 observed in both tumor and normal cells. In contrast, IR 60 activation of p44/42MAPK was observed only in tumor 50 IR + Caffeine cells but not in normal cells. However, activation of p44/ 42MAPK appeared to be associated with cytoprotective 40 50 30 functions as previously reported, since treatment with a 20 combination of Ad-PTEN, caffeine, and MEK1 inhibitor % Apoptotic Cell Number 10 UO126 resulted in lower expression of p44/42MAPK and 0 a significantly higher apoptotic rate than treatment with Days 3 Days 5 Ad-PTEN and caffeine only. Alternatively, the increased Days 3 Days 5 PTEN protein expression observed in tumor cells, but not PBS 2.54 5.465 in normal cells, when treated with Ad-PTEN and caffeine Ad-PTEN 6.135 26.95 could be responsible for the observed synergistic inhibi- Ad-PTEN + Caffeine 10.95 99 IR 7.01 18.4 tory effect. Whatever the underlying mechanism maybe, IR + Caffeine 9.155 15.45 the present study demonstrates that the combination treatment of Ad-PTEN and caffeine exerts synergistic c inhibitory effect in tumor cells but not in normal cells. To further test whether the effect and the function of PTEN as an growth inhibitor and apoptotic inducer are similar IR + Caffeine PBS Caffeine IR U0126 to other agents, HCT116 p53 ðþ=þÞ cells were treated with Ad-PTEN or IR. Cell-cycle analysis showed that IR p44/42 MAPK treatment induced G2/M arrest and caffeine abrogated the G2/M arrest similar to that observed with Ad-PTEN and β-actin caffeine treatment. However, associated with the abroga- 1.0 1.12 Dens. Ratio tion of the G2/M arrest, an increase in the number of apoptotic cells was not observed. This is due to the fact Figure 6 Effect of treatment with combinations of IR and caffeine. that the cell-cycle analysis was performed at early time HCT116 Á p53 ðþ=þÞ colorectal cancer cells were treated with PBS, points (48 hours). Analysis at later time points demon- Ad-PTEN, a combination of Ad-PTEN and caffeine, 2 Gy IR, a strated increased number of apoptotic cells (data not combination of IR and caffeine, or 10 mM U0126. (a) At 72 hours after shown). A major difference between PTEN and IR treatment, isobolograms at IC50 levels were generated for cells treated with the combination of IR and caffeine. (b) The numbers of treatment, however, was in the expression of p44/ cells at sub-G0/G1 (apoptotic cells) were analyzed by flow cytometry 42MAPK. IR induced p44/42MAPK expression but Ad- on days 3 and 5 after treatment. Data represent the means of PTEN did not, while caffeine enhanced its expression. duplicate experiments. Error bars denote standard error (SE). (c) At These results suggest that PTEN has an inhibitory effect 48 hours after treatment, cells were harvested and phosphorylation on phospho-p44/42MAPK, but IR does not, and caffeine status of p44/42MAPK examined by Western blot analysis. The may enhance this kinase activity because of cytoprotective corresponding b-actin levels are shown as a loading control. functions and/or mitogenic or differentiation-related stimuli.51,52 Furthermore, combined treatment with IR and caffeine produced an additive effect, but not synergy,

Cancer Gene Therapy PTEN and caffeine synergism in colorectal cancer cells Y Saito et al 812 and induced significantly fewer apoptotic cells than growth arrest in glioma cells. Cancer Res. 1998;58: treatment with Ad-PTEN and caffeine on day 5 after 5002–5008. treatment. These results suggest that PTEN is a more 9. Li DM, Sun H. PTEN/MMAC1/TEP1 suppresses the effective therapeutic agent when combined with caffeine. tumorigenicity and induces G1 cell cycle arrest in human In summary, the present study demonstrates that treat- glioblastoma cells. Proc Natl Acad Sci USA. 1998;95: ment with a combination of Ad-PTEN and caffeine 15406–15411. 10. Minaguchi T, Mori T, Kanamori Y, et al. Growth produces a synergistic therapeutic effect and selectively suppression of human ovarian cancer cells by adenovirus- induces apoptosis in colorectal cancer cells, especially in mediated transfer of the PTEN gene. Cancer Res. cancer cells that express wt-PTEN, but not in normal 1999;59:6063–6067. cells. 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