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Keywords:

  • perifosine;
  • curcumin;
  • colorectal cancer;
  • apoptosis;
  • signal transduction

Abstract

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Our study shows that coadministration of curcumin and an orally bioactive alkylphospholipid perifosine results in a significant increase in colorectal cancer cell apoptosis and a marked inhibition of cell growth both in vitro and in vivo. This novel combinatorial regimen leads to changes of multiple cell signaling pathways including inactivation of Akt and nuclear factor-κB as well as activation of c-Jun N-terminal kinases and endoplasmic reticulum stress. Further, perifosine and curcumin synergistically increase intracellular level of reactive oxygen species and ceramide, and downregulate the expression of cyclin D1 and Bcl-2 in colorectal cancer cells. These changes at molecular level together account for the cancer cell apoptosis and growth inhibition. We conclude that perifosine sensitizes colorectal cancer cells to curcumin by modulating multiple signaling pathways. Adding perifosine with curcumin may represent an effective therapy regimen against colorectal cancers, and possible other aggressive tumors.

Colorectal cancer is the second leading cause of cancer-related deaths in the United States. Annually, more than 56,000 newly diagnosed colorectal cancer patients die.1, 2 Curcumin has demonstrated anti-proliferative, anti-oxidant, anti-inflammatory, anti-angiogenic and many other anti-tumor effects.3, 4 The anti-tumor potential of curcumin relays on its ability to inhibit cancer cell proliferation and to induce cell apoptosis.5–7 Epidemiological studies suggest that curcumin uptake might be associated with the low rate of colorectal cancer in certain countries.2 Curcumin has been shown to induce apoptosis in human colorectal cancer cells both in vitro and in vivo; however, a relative high concentration of curcumin is needed to achieve its anti-tumor effects.3 Studies have shown that a combination of curcumin with traditional chemotherapeutics may provide a better strategy for the treatment of colorectal cancers, these chemotherapeutics include capecitabine,8 sulindac sulfone,3 5-fluorouracil9, 10 and oxaliplatin.9, 11

Perifosine is the first orally bioactive alkylphospholipid and has shown anti-tumor activities in multiple cancer cells and in preclinical animal models.12–14 Perifosine is currently in Phase II clinical trials.13, 15 The cellular targets of perifosine have not been fully understood although perifosine is known to block Akt activation16, 17 and to induce c-Jun NH2-terminal kinase (JNK) activation.18–21 Recent studies have also suggested that perifosine induces cell-cycle arrest in a p21-dependent manner.22 Most recently, perifosine is shown to increase cellular level of ceramide and reactive oxygen species (ROS)23, 24 and to inhibit extracellular signal-regulated kinases activation.23 Perifosine is able to attenuate chemoresistance and sensitize multiple cancer cells to traditional chemotherapy agents such as histone deacetylase inhibitors (HDACi), doxorubicin, paclitaxel and many others.12–14, 23, 24 However, the potential role of perifosine in colorectal cancers and its ability to sensitize curcumin are not well studied. Here, we show that perifosine sensitizes colorectal cancer cells to curcumin-induced apoptosis and growth inhibition by modulating multiple signaling pathways.

Material and Methods

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Chemicals and reagents

Perifosine was obtained from Shanghai Biochempartner (Shanghai, China) and was dissolved in PBS. Curcumin, fumonisin B1 and NAC were purchased from Sigma (St. Louis, MO). C6 Ceramide was obtained from Avanti (Alabaster, Alabama). SP600125 was obtained from Calbiochem (Natick, MA). Salubrinal, anti-Bcl-2, anti-cyclin D1, anti-AKT1/2, anti-CHOP, goat anti-rabbit IgG-HRP and goat anti-mouse IgG-HRP antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Monoclonal mouse anti-β-actin was obtained from Sigma (St. Louis, MO). All phosphorylation antibodies and anti-cleaved caspase 3, anti-cleaved poly(ADP–ribose) polymerase (PARP) antibodies were obtained from Cell Signaling Technology (Beverly, MA).

Cell culture

Colorectal cancer lines HT-29, HCT-116 and Caco2, pancreatic cancer cell line PANC-1, melanoma cell line WM-115 and glioblastoma cell line U87MG (obtained from Shanghai Maisha Biological Technology, Shanghai, China) were maintained in a Dulbecco's modified Eagle's medium (DMEM) (Sigma, St. Louis, MO), supplemented with a 10% fetal bovine serum (FBS) (Sigma, St. Louis, MO), Penicillin/Streptomycin (1:100, Sigma, St. Louis, MO) and 4 mM L-glutamine (Sigma, St. Louis, MO), in a CO2 incubator at 37°C.

Primary colon cancer cell isolation and culture

The fresh malignant colonic mucosal specimens from patients were thoroughly washed in PBS containing 100 unit/mL penicillin–streptomycin and 2 mM dithiothreitol (wash buffer) to remove debris and then minced by scalpel into small pieces into DMEM containing 100 unit/mL penicillin–streptomycin. Colon cancer cell pellets were thoroughly washed, then repelleted at 400g for 5 min. Single-cell suspensions of malignant colon cancer cells were achieved by resuspending cells in 0.15% (w/v) collagenase dissolved in DMEM and incubating the suspension at 37°C and 5% CO2. After 1 hr, individual cell was pelleted and rinsed twice with DMEM before resuspending the cell pellets in cell culture medium (DMEM, 15% FBS, 10 mg/mL transferrin, 2 mM glutamine, 1 mM pyruvate, 10 mM HEPES, 100 unit/mL penicillin/streptomycin, 0.1 mg/mL gentamicin, 0.2 unit/mL insulin, 0.1 mg/mL hydrocortisone and 2 g/L fungizone).

Western blots

Cells with indicated treatments were lysed with lysis buffer.25, 26 30 μg of protein of each sample was separated by 10% SDS–polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Millipore, Bedford, MA). After blocking with 10% milk, membranes were incubated with specific primary antibodies overnight at 4°C followed by incubation with secondary antibodies for 1 hr at room temperature. Antibody binding was detected with the enhanced chemiluminescence detection system.

Cell viability assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide dye assay)

Cell viability was measured by the 3-(4,5-dimethylthylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) method as reported earlier.25, 26

Assessment of the percentage of apoptotic cells

The In Situ Cell Death Detection Fluorescein Kit (Roche, Palo Alto, CA) was used to detect HT-29 cell apoptosis after indicated treatments according to the manufacturer's protocol. Briefly, the DNA fragments from the cells with different treatments were stained by TUNEL fluorencein, TUNEL-positive stained cells were detected using a regular fluorescence microscope. For each apoptosis experiment, at least 100 cells in five random scope fields were counted for apoptotic percentage. The percentage of apoptotic cells was calculated by total number of TUNEL-positive cells divided by the total number of the nuclear (stained by Hoechst 33342).

Quantification of apoptosis by ELISA

As reported earlier,25, 26 the Cell Apoptosis ELISA Detection Kit (Roche, Palo Alto, CA) was used to detect apoptosis in HT-29 cells with different treatments according to the manufacturer's protocol. Briefly, after indicated treatments, the cytoplasmic histone/DNA fragments from cells were extracted and bound to immobilized anti-histone antibody. Subsequently, the peroxidase-conjugated anti-DNA antibody was used for the detection of immobilized histone/DNA fragments. After addition of substrate for peroxidase, the spectrophotometric absorbance of the each sample was determined by using Dynatech MR5000 plate reader at 405 nM.

Caspase 3 activity assay

To evaluate caspase-3 activity, HT-29 cell lysates after each treatment were prepared according to the manufacturer's protocol (Promega, WI). Assays were performed in 96-well microtiter plates by incubating 20 μg cell lysates in 100 μL reaction buffer (1% NP-40, 20 mM Tris–HCl, pH 7.5, 137 mM NaCl, 10% glycerol) containing the caspase 3 substrate (DEVD-pNA) at 5 μM. Lysates were incubated at 37°C for 2 hr. Thereafter, the absorbance at 405 nm was measured with a Dynatech MR5000 spectrophotometer.

shRNA and plasmids transfection

shRNAs for AKT1/2 and PERK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A plasmid encoding a constitutively active AKT1 cDNA (Plasmid 16244) was purchased from Addgene (Cambridge, MA). HT-29 cells were seeded 2 days prior to transfection and cultured to 70% confluence before transfection. A Lipofectamine 2000 transfection method was used to transfect HT-29 cells according to the manufacturer's protocol (Invitrogen, CA). Successfully transfected cells were selected by puromycin (Sigma, Shanghai, China) for the shRNA plasmid. Western blots were performed to test transfection efficiency.

ROS production detection

As reported earlier,25, 26 cultured HT-29 cells were loaded with 1 μM fluorescent dye dihydrorhodamine 2 hr before treatment, which reacts with ROS in cells and results in a change of fluorescence. After indicated treatments, HT-29 cells were trypsinized, suspended in ice-cold PBS, and fixed in 70% ethyl alcohol in −20°C. The changes in fluorescence in drug-treated cells were quantified by FACS analysis. Induction of ROS generation was expressed in arbitrary units (vs. untreated control).

Ceramide generation detection

Ceramide accumulation in HT-29 cells after indicated treatments was measured using methods mentioned in Ref.27 and expressed as fold change vs. untreated control.

HCT-116 cell tumor xenograft in mice

Female SCID mice, 6 weeks old, were maintained with water and standard mouse chow ad libitum and used in protocols approved by the Nanjing Medical University's Animal Studies Committee. In brief, 1 × 106/0.1 mL of HCT-116 cells were injected subcutaneously by a 27-gauge needle into the right flanks of each mouse. After the tumor was established (50–75 mm3), as determined by caliper measurements (15th day after cancer cell injection), the mice were randomized into the following four groups (n = 5): (i) vehicle control; (ii) only curcumin-500 mg/kg by gavage daily for 30 days; (iii) perifosine-25 mg/kg body weight administered by gavage daily for 30 days and (iv) curcumin plus perifosine, following a similar schedule as described for individual drug treatment. The rationale for using the current doses of curcumin and perifosine were based on the observations by others that similar doses of these agents caused a significant reduction in tumor growth in mice,19, 28 tumor size were measured every 10 days by the modified ellipsoid formula: (π/6) × AB2, where A is the longest and B is the shortest perpendicular axis of an assumed ellipsoid corresponding to tumor mass. The body weight of mice in each group was also measured. At the end of treatment, the animals were sacrificed, and the tumors were freshly removed, then minced by scalpel into small pieces, solubilized and lysed in lysis buffer, followed by Western blots detecting signal proteins.

Statistical analysis

Data are presented as mean ± SD. Comparisons between groups were made with the paired Student's t-test. Values of p < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Perifosine sensitizes curcumin-induced colorectal cancer cell death

We first tested perifosine's effect on curcumin-induced colorectal cancer cell death. As shown in Figure 1a, at the concentrations we tested here (0.5, 1 and 5 μM), perifosine as a single agent had a minor effect on HCT-116 cell death (cancer cell death was reflected by reduced cell viability OD). Interestingly, perifosine dramatically sensitized curcumin-induced HT-29 death (Figs. 1a and 1b). Curcumin at 25 μM plus perifosine at 1 μM caused most obvious synergistic effect. In total, 25 μM of curcumin inhibited HT-29 cell viability by 17%, 1 μM of perifosine inhibited cell viability by 20%, whereas the combination of these two caused a synergistic 64% loss of cell viability (Figs. 1a and 1b). The sensitization effect of perifosine on curcumin was also seen in two additional colorectal cancer cell lines HCT-116 and Caco2 (Fig. 1c) and in primary cultured colon cancer cells (Fig. 1d). Further, perifosine also significantly enhanced cell death caused by clinically relevant chemotherapy agents, such as paclitaxel (Taxol), HDACi Trichostatin A and doxorubicin (Supporting Information Fig. S1A). Notably, the chemosensitization effect of perifosine on curcumin was also seen in the malignant glioblastoma cell line U87MG (Supporting Information Figs. S1B and S1C), ovarian cancer cell line SKOV3 (Supporting Information Figs. S1D and S1E), and in the pancreatic cancer cell line PANC-1 (Supporting Information Figs. S1F and S1G).

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Figure 1. Perifosine sensitizes colorectal cancer cell to curcumin-induced cell death. Serum starved human colorectal cancer cell lines HT-29 (a,b), HCT-116 and Caco-2 (c) and primary cultured colon cancer cells (cultured in a eight-well chamber, 2 × 104 in 0.5 mL/well) (d) were treated with a combination of indicated concentration of curcumin and perifosine, cell viability was measured using MTT assay 48 hr after treatment as measured, cell death was reflected by loss of cell viability compared to untreated control (ac). For primary cultured colon cancer cells, trypan blue was used to stain remaining live cells after indicated treatments (d). Loss of cell viability was calculated as follows: Loss of cell viability (%) = (MTT OD value of cells without treatment—MTT OD value of cells with indicated treatment(s))/MTT OD of cells without treatment. Experiments were repeated three times to insure consistency of results (Same for all the figures). The values in the figures are expressed as the means ± standard deviation (SD) (Same for all the figures). *p < 0.01 (Student's t-test) (Same for all the figures). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Perifosine sensitizes curcumin-induced colorectal cancer cell apoptosis and growth inhibition

Results in Figure 1 show that perifosine significantly increased curcumin-induced cancer cell viability loss/cell death, and then we tested whether this was associated with cell apoptosis. By using three different apoptosis assays including TUNEL staining, Histone-DNA ELISA and caspase-3 activity assay, we demonstrated that perifosine significantly increased curcumin-induced HT-29 cell apoptosis (Figs. 2a–2c). Western blot results in Figures 2d and 2e show that perifosine facilitated curcumin-induced caspase-3 and its downstream PARP cleavage (also see caspase 3 cleavage in HCT-116 cells [Supporting Information Figs. S2A and S2C]. Further, perifosine also enhanced curcumin-induced cell apoptosis in primary cultured colon cancer cells (Fig. 2g) and in two other colon cancer cell lines HCT-116 and Caco-2 (Supporting Information Fig. S1H). Importantly, Z-DEVDfmk, known as a specific caspase 3 inhibitor, largely inhibited perifosine and curcumin coadministration-induced HT-29 cell viability loss (Fig. 2f), suggesting that caspase-3 activation and apoptosis might be critical for cell viability loss/cell death by the coadministration in colorectal cancer cells. We then tested the role perifosine on curcumin-induced growth inhibition in colorectal cancer cells. As shown in Figures 2h and 2i, HT-29 cells that received both perifosine and curcumin treatments showed a significant inhibition of cancer cell proliferation compared to the cells that received single treatment of perifosine or curcumin. It is noted that caspase-3 inhibitor Z-DEVDfmk, which by itself had no detectable effect on cancer cell proliferation (data not shown), significantly suppressed growth inhibition by perifosine and curcumin coadministration in (Fig. 2h), suggesting that cell apoptosis and caspase-3 activation might be the key factor contributing to the slow cell growth in perifosine plus curcumin-treated cells.

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Figure 2. Perifosine sensitizes curcumin induced colorectal cancer cell apoptosis and growth inhibition. Serum starved HT-29 cells were either left untreated or treated with curcumin (25 μM), perifosine (1 μM), or a combination of both for indicated time points, cell apoptosis was measured by TUNEL staining (a) and Histone-DNA ELISA assay(c), caspase-3 activity in those cells was also measured (b). Expression level of cleaved caspase-3 and PARP was measured by Western blots (d) and were quantified in (e). Same treatments were also applied to primacy cultured colon cancer cells, and TUNEL staining was used to test cell apoptosis (g). The effect of caspase-3-specific inhibitor Z-DEVDfmk (50 μM) on perifosine (1 μM) plus curcumin (25 μM)-induced HT-29 cell death was shown in (f). HT-29 cells (10 × 103) were cultured in 15 cm-cultured dishes and maintained in 10% FBS DMEM medium with or without indicated drugs, cell growth was measured by cell number count (h) or MTT assay (i). Western blot results were quantified by Image J software after normalization to the loading controls and were expressed as fold changes vs. untreated control. *p < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Perifosine and curcumin synergistically block Akt/mTORC1 activation; inhibit IκBα phosphorylation and downregulate Bcl-2 and cyclin D1 expression

Activation of Akt is one of the key factors contributing chemoresistance in colorectal cancer and Akt is a main target of perifosine.18, 19, 22, 29 We then tested the effect of perifosine and curcumin on Akt activation in colorectal cancer cells. Western blots results demonstrated that 25 μM of curcumin was not able to inhibit Akt and downstream mTORC1 activation, whereas perifosine blocked them in both HT-29 cells (Figs. 3a and 3b) and HCT-116 cells (Supporting Information Figs. S2A and S2B). Curcumin is known to inhibit nuclear factor-κB (NF-κB) activation and to suppress the expression of NF-κB response gene products such as Bcl-2 and cyclin D1.8, 30–32 As PI3K/Akt activation is a known upstream signal molecular for NF-κB in a number of stimuli,33–35 and perifosine blocks Akt activation, we then tested perifosine's effect on NF-κB activation (IκBα phosphorylation) and expression of Bcl-2 and cyclin D1. Western blot results in Figures 3a and 3b show that either curcumin or perifosine alone treatment suppressed IκBα phosphorylation and Bcl-2/cyclin D1expression in HT-29 cells, combination of these two caused a profound inhibition of IκBα phosphorylation and downregulation of Bcl-2 and cyclin D1 in colorectal cancer lines HT-29 cells (Figs. 3a and 3b) and HCT-116 cells (Supporting Information Figs. S2A–S2C), and in primary cultured colon cancer cells (Fig. 3c). Interestingly, shRNA-mediated Akt1/2 knockdown aggravated curcumin induced the pIκBα inhibition and Bcl-2/cyclin D1 downregulation, as well as cancer cell apoptosis and viability loss (Fig. 3e and 3f, Supporting Information Figs. S3A and S3B) to suggest that Akt activation serves as upstream signal for IκBα phosphorylation. On the other hand, exogenously introducing a constitutively active Akt restored Akt activation and cell viability in curcumin and perifosine coadministration-treated HT-29 cells (Figs. 3g and 3h), indicating that Akt inhibition is involved in cancer cell viability loss by the coadministration.

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Figure 3. Perifosine and curcumin synergistically inhibit Akt/mTORC1 activation, reduce IκBα phosphorylation and downregulate Bcl-2/Cyclin D1 expression. HT-29 cells were either left untreated or treated with curcumin (25 μM), perifosine (1 μM) or a combination of both for 12 hr, Akt and downstream mTORC1 activation, IκBα phosphorylation (Ser 32), cyclin D1 and Bcl-2 expressions and indicated loading controls were tested by Western blots using indicated antibodies (a). Akt and IκBα phosphorylation, as well as cyclin D1 and Bcl-2 expression levels were quantified in (b). Primary cultured colon cancer cells were left untreated (Ctrl) or treated with curcumin (25 μM) plus perifosine (1 μM), Akt and IκBα phosphorylation and expression level of Bcl-2 and cyclin D1 were tested (c). HT-29 cells were transfected with Akt1/2 shRNA, successfully transfected cells were selected by puromycin, expression level of Akt after transfection was tested to confirm transfection efficiency. Effects of Akt1/2 knockdown on IκBα phosphorylation (Ser 32), cyclin D1 and Bcl-2 expression (d) as well as cancer cell death (e) and apoptosis (f) in curcumin (25 μM)-treated HT-29 cells are shown. Using the same transfection protocol, HT-29 cells were transfected with constitutively active (CA)-Akt before adding curcumin (25 μM) plus perifosine (1 μM) (Cur + Prf), Akt phosphorylation (12 hr after the treatment) and cell death (48 hr after the treatment) were detected in (g) and (h), respectively. Experiments were repeated at least three times to insure consistency of results. The values in the figures are expressed as the means ± standard deviation (SD). *p < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Perifosine increases curcumin-induced ROS, ceramide production and JNK activation

Previous studies have shown that curcumin induces a robust ROS and ceramide production to mediate cancer cell apoptosis,27 further, exogenously adding short-chain ceramide (C6) sensitizes melanoma cell to curcumin.36 Interestingly, recent studies have shown that perifosine is able to enhance HDACi-induced ROS and ceramide production.23 Further, Sun et al. show that perifosine increases ceramide production to sensitize taxol-mediated ovarian cancer cell apoptosis.24 Here, we found that perifosine enhanced curcumin-induced ROS and ceramide production in HT-29 cells (Figs. 4a and 4b). The antioxidant NAC, which reduced curcumin and perifosine coadministration-induced ROS production (Fig. 4a), also inhibited ceramide accumulation (Fig. 4c), suggesting that ROS production is important for ceramide accumulation after the coadministration. Both fumonisin B1,37 a de novo ceramide synthesis inhibitor, and the antioxidant NAC inhibited curcumin and perifosine coadministration-induced HT-29 cell death and apoptosis (Fig. 4d) as well as primary colon cancer cell death (Fig. 4e). These data together suggest that perifosine facilitates curcumin-induced ROS production to increase intracellular ceramide level and cancer cell apoptosis. Previous study has shown that curcumin induces ROS/ceramide production to activate proapoptotic JNK in colon cancer cells.6, 27 Western blot results in Figure 4f confirm that JNK activation (JNK1/2 and cJun phosphorylation) after curcumin treatment in HT-29 cells. Perifosine, which by itself induced a moderate JNK activation, dramatically enhanced curcumin-induced JNK activation (Fig. 4f). The fact that NAC or fumonisin B1 largely inhibited perifosine and curcumin coadministration-induced JNK activation suggests that ROS and ceramide are upstream signals for JNK activation (Fig. 4g). Importantly, the JNK inhibitor SP 600125 inhibited curcumin and perifosine coadministration induced cell death and apoptosis in HT-29 cells (Figs. 4h and 4i) and in primary cultured colon cancer cells (Fig. 4k). Western blot results in primary cultured colon cancer cells confirmed JNK activation after curcumin and perifosine coadministration (Fig. 4j). These data suggest that ROS/ceramide-dependent activation of JNK by curcumin and perifosine coadministration is involved in cancer cell apoptosis.

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Figure 4. Perifosine increases curcumin-induced ROS, ceramide production and JNK activation HT-29 cells were either left untreated or treated with curcumin (25 μM), perifosine (1 μM) or a combination of both, ROS and ceramide production were shown in (a) and (b), respectively. The role of fumonisin B1 (20 μM) or NAC (400 μM) on curcumin + perifosine-induced ceramide production and cell death/apoptosis in HT29 cells are shown in (c) and (d), respectively. (e) Fumonisin B1 (20 μM) or NAC (400 μM) inhibited curcumin + perifosine-induced primary colon cancer cell death. HT-29 cells were either left untreated or treated with curcumin (25 μM) with or without perifosine (1 μM) for indicated time points, JNK and Jun phosphorylation were detected (f). The role of fumonisin B1 (20 μM, 1-hr pretreatment) or NAC (400 μM, 1-hr pretreatment) on curcumin + perifosine-induced JNK and Jun phosphorylation are shown in (g). Effects of JNK inhibitor SP 600125 (10 μM, 1-hr pretreatment) on curcumin (25 μM) + perifosine (1 μM)-induced cell death and apoptosis are shown in (h,i) (HT-29 cells) and (k) (primary colon cancer cells). JNK activation after curcumin (25 μM) + perifosine (1 μM) treatments in primary colon cancer cells are shown in (j). *p < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Perifosine aggravates curcumin-induced endoplasmic reticulum stress

Previous studies have shown that curcumin induces endoplasmic reticulum stress response, which is important for cancer cell apoptosis.38, 39 Inhibition of endoplasmic reticulum stress (ER stress) by RNAi-mediated CHOP downregulation or by its inhibitor salubrinal40 significantly reduces curcumin-induced cell apoptosis.39 Western blot results in Figure 5a here confirmed an obvious ER stress response (PERK, eIF2α phosphorylation and CHOP expression) after curcumin treatment in HT-29 cells, and perifosine aggravated curcumin-induced ER stress response (Fig. 5b). ER stress inhibitor salubrinal reduced perifosine and curcumin coadministration-induced HT-29 cell death (Fig. 5c) and ROS production (Fig. 5d). Further, shRNA knockdown of PERK inhibited coadministration-induced ER stress response, cell death and apoptosis (Figs. 5e and 5f, Supporting Information Figs. S3A and S3C). Data in Figure 5g confirm that ER stress response after perifosine and curcumin treatment in primary colon cancer cells, and salubrinal reduced perifosine and curcumin coadministration-induced primary colon cancer cell death (Fig. 5h), these data together suggest that perifosine aggravates curcumin-induced ER stress, which is important for cell apoptosis.

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Figure 5. Perifosine aggravates curcumin-induced ER stress. HT-29 cells were either left untreated or treated with curcumin (25 μM) with or without perifosine (1 μM) for indicated time points, ER stress response was measured by detecting PERK, eIF2α phosphorylation and CHOP expression (a,b). Effects of salubrinal on curcumin (25 μM)+perifosine (1 μM)-induced cell death and ROS production are shown in (c) and (d), respectively. HT-29 cells were transfected with PERK shRNA, successfully transfected cells were selected by puromycin. Stable PERK knockdown HT-29 cells were confirmed by Western blots detecting PERK level (e). Effects of PERK knockdown on curcumin (25 μM)+perifosine (1 μM) induced ER stress response, cell death and apoptosis are shown in (e,f). Primary cultured colon cancer cells were treated with curcumin (25 μM)+perifosine (1 μM) for indicated time points, ER stress response is shown in (g). Primary colon cancer cells were either left untreated or pretreated with salubrinal (10 μM) for 1 hr, followed by curcumin (25 μM) plus perifosine (1 μM) exposure for another 48 hr, trypan blue was then used to stain remaining survival cells. The number of trypan blue-positive cells in treatment groups was recorded and was normalized to the number of cells in untreated group (h). *p < 0.01. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Curcumin together with perifosine causes significant inhibition of colon cancer cell growth in vivo

The above in vitro results strongly support an efficient killing of colorectal cancer cells by the novel combinatorial regimen of curcumin and perifosine. To further validate our in vitro results, we compared the effectiveness of combination therapy with monotherapy on the growth of HCT-116 cells in mice xenograft colon cancer model in SCID mice. It was observed that curcumin (body weight, 500 mg/kg) and perifosine (body weight, 25 mg/kg), each alone, inhibited the HCT-116 tumor growth by 21% (p < 0.01) and 30% (p < 0.01), respectively, compared to the controls (Fig. 6a). Significantly, the combination of curcumin and perifosine produced a significant 54% inhibition (p < 0.05) in tumor growth relative to the control, demonstrating enhanced inhibitory effect of the combination therapy in the colon cancer xenograft model (Fig. 6a). None of these treatments had any effect on body weight (Fig. 6b) and diet consumption (data not shown) during the 30 days of treatments. No other signs of systemic toxicity or any adverse effects as monitored by activity and posture of mice were observed, suggesting that neither curcumin nor perifosine alone, or in combination caused any deleterious effects under the present experimental conditions. Western blot results in Figure 6c confirm that signal changes observed in the in vitro experiments, and curcumin together with perifosine coadministration largely inhibited Akt/NF-κB activation, downregulated Bcl-2/Cyclin D1 expression, and induced PERK/JNK phosphorylation in vivo.

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Figure 6. Curcumin together with perifosine causes significant inhibition of colon cancer cell growth in vivo (a) Coadministration of curcumin (500 mg/kg body weight, p.o.) and perifosine (25 mg/kg body weight, p.o.) caused a significant inhibition of tumor growth in a mice colon cancer xenograft model; this is associated with signal changes in (c). Mice body weight after corresponding treatments was also recorded (b). *p < 0.01 vs. vehicle control group. **p < 0.01 vs. curcumin only group. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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Discussion

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Recent studies have suggested that inclusion of curcumin in conventional chemotherapeutic regimens could be an effective strategy to prevent the emergence of chemoresistant in colon cancer cells41 and coadministration of curcumin and FOLFOX greatly reduced the survival of colon cancer cells42, 43 and cancer stem cells,43 associated with a concomitant reduction in activation of multiple growth factor receptors and downstream survival signals, as well as the expression of cyclin-D142. Coadministration of curcumin and resveratrol also caused a synergistic inhibition of colon cancer.28 Here, we found that perifosine significantly enhanced curcumin-induced apoptosis in colorectal cancer cell lines and in primary colon cancer cells derived from human patients, and was correlated with a increased activity of caspase-3, an enhanced ROS and ceramide accumulation, an overactivated JNK signaling and a profound ER stress response. Coadministration curcumin and perifosine also synergistically inhibited Akt and NF-κB activation and downregulated Bcl-2 and cyclin D1 expressions. These signal changes together contributed to colon cancer cells apoptosis.

Bioavailability of curcumin is generally poor after oral administration.44 Phase I clinical trials have shown that oral doses of curcumin at 8,000 mg/day produced peak serum curcumin levels of only 1.77 ± 1.87 μM.44 Also, the data from the same study suggest that higher curcumin concentrations achieved in intestine and colon after oral administration, which might be associated with greater biological activity and better chemopreventive effectiveness in those tissues than in other organs.44 However, a relative high concentration of curcumin is still needed to achieve its anti-tumor ability.6 Previous study and our own data here suggest that curcumin at concentration of 25–30 μM did not result in a significant cell death and apoptosis in vitro,6 In our study, we found that a relative low dose of curcumin (12.5–25 μM) can cause significant cancer cell apoptosis in the presence of perifosine.

It has been demonstrated that Akt signaling can activate the NF-κB signaling under of a number of stimuli, such as tumor necrosis factor (TNF-α), platelet-derived growth factor and many others.34, 45, 46 In our study, we found that abolishing Akt phosphorylation by perifosine or knocking down Akt by specific shRNA although reduced (∼50% inhibition), but not completely blocked the IκB phosphorylation in both HT-29 cells (Figs. 3a, 3b and 3d) and HCT-119 cells (Supporting Information Fig. S2), indicating that Akt along with other unidentified upstream molecular contribute to fully activation of NF-κB in these cells. The fact that 25 μM of curcumin inhibited the IκB phosphorylation while leaving Akt phosphorylation unchanged suggests that curcumin's effect on IκB phosphorylation is Akt independent, and may be through inhibiting the unidentified upstream molecular (Figs. 3a, 3b and 3d; Supporting Information Fig. S2), this could also explain the synergistic inhibitory effect on IκB phosphorylation by curcumin plus perifosine treatment, as together they not only blocked Akt activation (by perifosine), but should also inhibit those unidentified upstream molecular (by curcumin).

Previous study has shown that curcumin can block cytokine- and phorbol ester-stimulated JNK activation, c-jun phosphorylation and AP-1 transcriptional activity.47 On this basis, curcumin has been used as an inhibitor of JNK. However, in colon cancer cells, studies have confirmed that curcumin actually promotes the activation of JNK and its downstream c-jun, and stimulates AP-1 transcriptional activity,6 and blockade of JNK activation by SP 600125 inhibits curcumin-induced apoptosis,6 suggesting that JNK activation is involved in curcumin-induced apoptosis. Here, we confirmed that JNK is activated by curcumin in HT-29 cells and in primary cultured colon cancer cells, perifosine enhanced curcumin-induced JNK activation to participate colorectal cancer cell apoptosis.

It is important to note that interference with JNK signaling or any other single signal pathways using the method described here did not result in total abolition cancer cell death/apoptosis by curcumin and perifosine coadministration. This may be owing to incomplete inhibition of any of the single pathways. More likely, however, is that these signaling events together contribute to colorectal cancer cell apoptosis. Western blot results in Supporting Information Figure S4 show that the antioxidant NAC, the ceramide inhibitor fumonisin B1 and ER stress inhibitor salubrinal (data not shown) had no obvious effect on perifosine and curcumin coadministration-induced Akt inhibition and Bcl-2/cyclin D1downregulation, suggesting that ROS/ceramide production and ER stress response are independent of Akt inhibition and Bcl-2/cyclin D1 downregulation by the coadministration.

References

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Material and Methods
  4. Results
  5. Discussion
  6. References
  7. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IJC_27548_sm_SuppFigS1.eps1385KSupporting Information Figure 1.
IJC_27548_sm_SuppFigS2.eps1577KSupporting Information Figure 2.
IJC_27548_sm_SuppFigS3.eps958KSupporting Information Figure 3.
IJC_27548_sm_SuppFigS4.eps1149KSupporting Information Figure 4.

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