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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

Anti-angiogenesis has been a promising strategy for cancer therapy. However, many signal pathways are activated during anti-angiogenic treatment to counteract the therapeutic efficacy. Among these pathways, evidence has directly pointed to the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway, whose activation resulted in tolerance to the absence of nutrients and oxygen when tumor angiogenesis has been inhibited. In the present study, we investigated the effects of blocking activation of the PI3K/Akt pathway on cell survival in vitro and tumor growth in vivo during anti-angiogenesis therapy. In modeled microenvironments in vitro, we observed that the phosphorylation of Akt in tumor cells was increased gradually in the absence of serum and oxygen in a time-dependent manner. The specific inhibitors of PI3K inhibited the proliferation of tumor cells in a dose-dependent manner in vitro. Moreover, inhibition was enhanced gradually with increased serum deprivation and/or hypoxia. In a mouse tumor model, we found the phosphorylation of Akt obviously increased following anti-angiogenic therapy using plasmids encoding soluble vascular endothelial growth factor receptor-2, but significantly reduced after treatment with LY294002. Consequently, the combinational treatment exhibited better antitumor effects compared with single treatments, presenting larger necrosis-like areas, more apoptotic cells, less microvessel density and less phosphorylated Akt in tumors. These results suggest that blocking activation of the PI3K/Akt pathway during anti-angiogenesis therapy could enhance antitumor efficacy. Thus, targeting the PI3K/Akt pathway might be a promising strategy to reverse tumor resistance to anti-angiogenesis therapy. (Cancer Sci 2011; 102: 1469–1475)

Angiogenesis is essential to tumor growth and metastasis, which is modulated by a number of receptors and growth factor ligands in the tumor microenvironment.(1) Among these receptors, vascular endothelial growth factor receptor-2 (VEGFR2) is one of the most important receptors that mediates angiogenesis through binding vascular endothelial growth factor (VEGF), as described in previous studies.(2,3) Furthermore, VEGFR2 not only has a more restricted expression on endothelial cells but also is upregulated once these cells proliferate during tumor neovascularization.(3) These characteristics of VEGFR2 have made it a promising target for anti-angiogenic therapy, and have already got a series of encouraging efficacies for cancer therapy with strategies of blocking VEGFR2, such as dominant-negative receptor mutants, recombinant soluble VEGFR2 (sVEGFR2), monoclonal antibodies against VEGFR2 and some synthetic inhibitors.(4–7)

Angiogenesis is indispensable to tumor growth and metastasis. During anti-angiogenesis therapy the cutting off of nutrients and oxygen with the decrease of microvessels brings many changes to the tumor microenvironment under metabolic stress.(8–10) These changes include local hypoxia, less nutrients and lower pH, and the activation of many signal pathways that promote cellular survival and growth for rapid adaptation to alterations of tumor microenvironments.(8,9,11) The phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway, frequently overactivated in many tumors but tightly regulated in normal cells, is one of the pathways being activated by growth factors, hypoxia and nutrient deficiency.(12–16) It is implicated in cellular signal regulation and plays important roles in inhibiting apoptosis, promoting proliferation, and inducing angiogenesis or formulation of vasculogenic mimicry in tumors.(8,17–21) Furthermore, it still causes tolerance of tumor cells to chemotherapy and radiotherapy, as well as targeting therapies such as epithelial growth factor receptor (EGFR) and insulin-like growth factor-1 receptor (IGF-IR) antagonism.(22) In particular, it causes tolerance to hypoxia, low nutrients and anti-angiogenesis.(8,15,20,23,24) These findings suggest that activation of the PI3K/Akt pathway largely attenuates the therapeutic efficacy by various mechanisms. Conversely, abolition of PI3K with inhibitor can not only induce apoptosis of tumor cells, but also increase sensitivity to chemotherapy, radiotherapy and targeting therapies.(22,25–28) More importantly, inhibiting PI3K still reduces the tolerance of tumor cells to hypoxia and nutrient deprivation in vitro.(15,24) Therefore, we further hypothesized that inhibiting PI3K might restore the sensitivity of tumor cells to anti-angiogenic treatment and improve its efficacy.

To test this hypothesis, we blocked activation of the PI3K/Akt pathway during treatments with serum deprivation and/or hypoxia in vitro or anti-angiogenic therapy with plasmids encoding sVEGFR2 in vivo. The results showed that blocking activation of the PI3K/Akt pathway significantly decreased cell survival in vitro and tumor growth in vivo during anti-angiogenesis therapy, indicating that targeting the PI3K/Akt pathway might be a promising strategy to reverse tumor resistance to anti-angiogenesis therapy.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

Cell lines and culture.  Murine cell lines of colon carcinoma CT26, Lewis lung carcinoma LL/2 and melanoma B16 were cultured in RPMI-1640 medium supplemented with 10% FBS and 100 μg/mL amikacin at 37°C with 5% CO2 atmosphere, and supplied with fresh medium every 2–3 days.

Detection of cellular PI3K/Akt activity in vitro.  For the detection of activity of the PI3K/Akt pathway we assessed the levels of phosphorylated Akt (on Ser473) with western blot in modeled tumor microenvironments through depriving serum and oxygen in vitro. Briefly, when cell confluences reached 50–60%, CT26 cells seeded in six-well plates were treated with 150 μM CoCl2 + 2% FBS for 0, 5, 8, 12, 24, 48 or 72 h, respectively. In the LY294002 dose-dependent inhibition assay, CT26 cells were treated with 0, 5, 10 or 20 μM LY294002 under normal or 150 μM CoCl2 + 2% FBS conditions. Each sample was separately detected with antibodies of anti-Akt, anti-Akt-S473 or anti-beta-actin (Cell Signaling Technology, Beverly, MA, USA).

Cell viability analysis by MTT assay.  Tumor cells were seeded into 96-well plates at 1–2 × 103/well, and then treated respectively as follows: first, CT26 cells were treated with LY294002 at 0, 5, 10 or 20 μM, respectively. Second, CT26 cells were treated with 10%, 2% or 0% FBS separately, and 10 μM LY294002 was added. Third, CT26 cells were treated with CoCl2 at 0, 50 or 100 μM, respectively, in combination with 10 μM LY294002. Fourth, CT26 cells were treated with 10% FBS + 0 μM CoCl2, 2% FBS + 50 μM CoCl2 or 0% FBS + 100 μM CoCl2, with 10 μM LY294002 simultaneously added to the above cells. In addition, all cells above with added LY294002 had a corresponding control, that is, only supplemented with the equivalent DMSO. Last, CT26, LL/2 or B16 cells were treated with 10% FBS, 2% FBS, 100 μM CoCl2, 10 μM LY294002 (and wortmannin for CT26 cells), 2% FBS + 10 μM LY294002 (and wortmannin for CT26 cells), 100 μM CoCl2 + 10μM LY294002 (and wortmannin for CT26 cells), 2% FBS + 100 μM CoCl2 or 2% FBS + 100 μM CoCl2 + 10 μM LY294002 (and wortmannin for CT26 cells), respectively. After all the cells above were incubated for 24 h, a MTT assay was carried out. Untreated cells acted as the indicator of 100% cell viability. The absorbance in each sample was measured at 570 nm by a Spectra MAX microplate reader (Spectra MaxL; Molecular Devices, Sunnyvale, CA, USA).

Preparation of plasmid.  Soluble VEGFR2, comprising a fragment of N-terminal 755 amino acid residues located in the extracellular domains, can directly neutralize VEGF and inhibit VEGF-induced angiogenesis.(5,8) It was amplified by PCR with forward primer 5′-AGCACCGGCGATGGAGAGCAAGGCGCTGCT-3′ and reverse primer 5′-CCGCTCGAGTTAGGCACCTTCTATTATGAAGAGC-3′ from pORF-mVEGFR2 (Invivogen, San Diego, CA, USA). Amplified products were then subcloned into the pVITRO2 expression vector (Invivogen). After sequence confirmation, the recombinant plasmid was prepared with Endofree Plasmid Giga kit (Qiagen, Chatsworth, CA, USA) and stored at –20°C.

Combination therapy in vivo.  Male BALB/c mice obtained from the West China Experimental Animal Center (Chengdu, China) were implanted subcutaneously with 1 × 106 CT26 cells in the right hind flanks. After 7 days, the tumor-bearing mice were randomly divided into four groups (six mice per group) and treated with pVITRO2-sVEGFR2, LY294002, pVITRO2-sVEGFR2 + LY294002 or PBS, respectively. The recombinant plasmid was injected intramuscularly with 50 μg/mouse, while the LY294002 was injected intratumorally with 7.5 mg/kg the following day. All treatments were performed every 3 days for five times. Tumor volumes were measured with calipers every 2 days and calculated according to 0.52 × length × width2. During treatment, mice were monitored closely and killed after 25 days when tumor cells were implanted.

Histological analysis and apoptosis detection.  Paraformaldehyde-fixed tumors from each group were embedded in paraffin and cut into 3–5 μm sections. Tumoral necroses were then evaluated with hematoxylin and eosin (HE) by the computer-aided image analysis system Quantimet 600 and Qwin software (Leica, Benshaim, Germany). Apoptotic cells in tumors were determined by the terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay (DeadEnd Fluorometric TUNEL System; Promega, Madison, WI, USA) according to the manufacturer’s guide.

Assessment of angiogenesis and phosphorylated Akt in vivo.  Angiogenesis and phosphorylation of Akt in tumors were determined by immunohistochemical analysis in frozen sections with anti-CD31 antibody (1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and in paraffin-embedded sections with anti-Akt-S473 antibody (Cell Signaling Technology), respectively. The microvessel density and phosphorylated-Akt ratios were identified respectively by counting the number of microvessels and phosphorylated Akt from five high-power fields.

Statistical analysis.  All results were analyzed statistically by one-way analysis of variance (anova) and unpaired Student’s t-test. All data were presented as means ± SD. The statistical significance was defined as P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

Activation of the PI3K/Akt pathway induced by Serum and oxygen deprivation.  We first tested the levels of phosphorylated Akt in CT26 cells in modeled tumor microenvironments (hypoxia and low serum) in vitro at various time. Western blot analysis revealed that phosphorylation of Akt increased gradually in a time-dependent manner in the absence of serum and oxygen compared with untreated cells, but that of total Akt remained invariable (Fig. 1A). The results indicated that the absence of serum and oxygen activated the PI3K/Akt pathway. Activation of the PI3K/Akt could be effectively blocked by LY294002, the selective inhibitor of PI3K/Akt, in a dose-dependent manner (Fig. 1B).

image

Figure 1.  Activation and inhibition of the phosphatidylinositol 3-kinase/Akt (PI3K/Akt) pathway in CT26 cells. CT26 cells were treated with 2% FBS + 150 μM CoCl2 for 0, 5, 8, 12, 24, 48 and 72 h (A), or were cultured under 2% FBS + 150 μM CoCl2 conditions and treated with LY294002 at 0, 5, 10 or 20 μM for 24 h, respectively (B). Western blot analysis was used to detect phosphorylated Akt (Akt-S473) protein, and beta-actin acted as a control.

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Effects of inhibiting PI3K in CT26 tumor cells in the absence of serum and/or oxygen.  Based on the above results, we hypothesized that activation of PI3K/Akt might endow cancer cells to acquire tolerance for serum and oxygen deprivation. Consequently, if activation of PI3K/Akt were blocked, the death of cancer cells would increase significantly. To validate this hypothesis, we first examined the effects of LY294002 on CT26 tumor cells under normal and serum/oxygen double-deficient conditions (CT26 cells were treated with 150 μM CoCl2 + 2% FBS). The results showed that the selective inhibitor of PI3K, LY294002, significantly decreased the survival of CT26 cells under normal and serum/oxygen double-deficient conditions in a dose-dependent manner (Fig. S1 and Fig. 2A).

image

Figure 2.  Effect of inhibiting PI3K with LY294002 on CT26 cells in the absence of serum and/or oxygen. MTT assays were performed to evaluate cell viability (untreated cells were defined as 100%). (A) CT26 cells were treated with LY294002 at 0, 5, 10 or 20 μM under 10% FBS + 0 μM CoCl2 or 2% FBS + 100 μM CoCl2 conditions for 24 h, respectively. (B) CT26 cells were cultured in 10%, 2%, 0% FBS medium, respectively, with or without 10 μM LY294002. (C) CT26 cells were treated with 0, 50 and 100 μM CoCl2, respectively, with or without 10 μM LY294002. (D) CT26 cells were treated with 10% FBS + 0 μM CoCl2, 2% FBS + 50 μM CoCl2 and 0% FBS + 100 μM CoCl2, respectively, with or without 10 μM LY294002. *< 0.05.

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The absence of serum and oxygen always varied from part to part in tumors.(29) To model various nutritional statuses of tumors, we treated CT26 cells with different media containing 10%, 2% or 0% FBS with or without 10 μM LY294002. Compared with cells that were cultured in normal conditions (10% FBS), the viability of cells under 2% or 0% FBS medium were 109.1 ± 8.2% or 78.3 ± 6.8%, respectively. Under media containing LY294002, cell viability decreased to 61.7 ± 7.0% and 43.1 ± 5.2%, respectively (P < 0.05; Fig. 2B). These results indicated that inhibition of PI3K obviously decreased cell survival with the increase of serum deprivation.

To model various levels of hypoxia in tumors, we treated CT26 cells with different media containing 0, 50 or 100 μM CoCl2 with or without 10 μM LY294002. Compared with control cells that were cultured in normal conditions (untreated cells), the viability of cells treated with 50 μM or 100 μM CoCl2 were 83.7 ± 6.9% or 72.2 ± 5.0%, respectively. Under media containing LY294002, cell viability decreased to 79.0 ± 7.3% (0 μM CoCl2 + 10 μM LY294002), 55.9 ± 4.9% (50 μM CoCl2 + 10 μM LY294002) and 49.3 ± 3.2% (100 μM CoCl2 + 10 μM LY294002), respectively (P < 0.05; Fig. 2C). These results indicate that inhibition of PI3K obviously decreased cell survival with the increase of hypoxia.

To model various statuses of hypoxia/nutrient double deficiency in tumors, we treated CT26 cells with different media containing 10% FBS + 0 μM CoCl2, 2% FBS + 50 μM CoCl2 or 0% FBS + 100 μM CoCl2, simultaneously with or without 10 μM LY294002. The results indicated that cell viability under 2% FBS + 50 μM CoCl2 or 0% FBS + 100 μM CoCl2 were 98.9 ± 9.0% or 45.6 ± 5.6%, respectively, while that under 10% FBS + 0 μM CoCl2 + 10 μM LY294002, 2% FBS + 50 μM CoCl2 + 10 μM LY294002, or 0% FBS + 100 μM CoCl2 + 10 μM LY294002 were 79.0 ± 7.3%, 39.0 ± 4.2%, or 16.4 ± 2.2%, respectively (P < 0.05; Fig. 2D). These findings suggest that inhibiting the PI3K pathway could decrease cell survival more significantly in more hostile tumor microenvironments.

Effects of inhibiting PI3K in various tumor cells in the absence of serum and/or oxygen.  To further validate the above results, we examined the cell viability of LL/2, B16 and CT26 cells, under the treatments different combinations of 10% FBS, 2% FBS, 100 μM CoCl2 and 100 μM LY294002, respectively (Fig. 3A–C). In all of these three kinds of tumor cells, the stimulus of 2% FBS alone could promote growth approximately 10%, while the single treatment of 100 μM CoCl2 or 10 μM LY294002 slightly inhibited cell growth approximately 20%. When two treatments were combined (2% FBS + 10 μM LY294002, 2% FBS + 100 μM CoCl2 or 100 μM CoCl2 + 10 μM LY294002), cell viability decreased to 50–70%. When activation of the PI3K pathway was inhibited and in a hypoxic and low-serum microenvironment (modeled by 2% FBS + 100 μM CoCl2), only one-third of tumor cells survived (Fig. 3A–C). To further confirm the observed effects of LY294002 were the result of PI3K inhibition, but not of other protein inhibition such as casein kinase-2,(30,31) we tested the effects of wortmannin, the other widely used PI3K inhibitor that is structurally unrelated to LY294002, on CT26 cells. The data showed that the inhibiting efficacy of wortmannin was similar to LY294002 in a modeled low-nutrient and hypoxic microenvironment (Fig. 3D). These results suggest that PI3K inhibition decreased the survival of various tumor cells in tumor microenvironments lacking serum and/or oxygen.

image

Figure 3.  Effect of inhibiting PI3K with LY294002 on various tumor cells in the absence of serum and/or oxygen. CT26 (A), LL/2 (B) or B16 (C) cells were treated with 10% FBS (control), 2% FBS, 100 μM CoCl2, 10 μM LY294002, 2% FBS + 10 μM LY294002, 100 μM CoCl2 + 10 μM LY294002, 2% FBS + 100 μM CoCl2 and 2% FBS + 100 μM CoCl2 + 10 μM LY294002, respectively. (D) CT26 cells were treated with 10% FBS (control), 2% FBS, 100 μM CoCl2, 10 μM wortmannin, 2% FBS + 10 μM wortmannin, 100 μM CoCl2 + 10 μM wortmannin, 2% FBS + 100 μM CoCl2 and 2% FBS + 100 μM CoCl2 + 10 μM wortmannin, respectively. All cells mentioned above were evaluated using MTT assay after 24 h. Untreated cells were defined as the indicator of 100% cell viability. *< 0.05.

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Blocking the PI3K pathway enhanced antitumor efficacy of anti-angiogenesis therapy.  To determine whether the antitumor capability could be augmented by blocking the PI3K/Akt pathway during anti-angiogenic treatment in vivo, the mice burdened with CT26 tumors were treated with PBS, LY294002, pVITRO2-sVEGFR2 and pVITRO2-sVEGFR2 + LY294002, respectively. As expected, a more significant inhibition of tumor growth (53.3%) was observed in mice treated with pVITRO2-sVEGFR2 combined with LY294002 (P < 0.05; Fig. 4A). In contrast, only limited antitumor efficacy was detected in mice treated with LY294002 (20.5%) or pVITRO2-sVEGFR2 (34.9%) alone (Fig. 4A). Accordingly, the average tumor weight in mice receiving combinational therapy was significantly lower than the control and single treatments (P < 0.05; Fig. 4B). Therefore, these results indicate that inhibition of the PI3K pathway enhanced the antitumor effect of anti-angiogenic therapy in vivo.

image

Figure 4.  Effect of combination therapy on CT26 tumor in a mouse model. The CT26-bearing mice (n = 6) were treated with pVITRO2-sVEGFR2, LY294002, pVITRO2-sVEGFR2 + LY294002 and PBS, respectively. The recombinant plasmid was injected intramuscularly with 50 μg/mouse, while LY294002 was injected intratumorally with 7.5 mg/kg. (A) Tumor growth was monitored once every 3 days and the volume was calculated as 0.52 × length × width2. (B) Tumors were isolated and weighed when mice were killed. *< 0.05.

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Increased apoptosis in tumors.  Pathological analysis through HE staining was performed to investigate histological changes in local tumor tissues. Compared with controls, lots of largely necrosis-like areas, characterized by nuclear fragmentation, cytoplasmic debris and obtrite cells, were observed in combination-therapy tumors (Fig. 5A). To validate whether cell death resulted from apoptosis, we detected the apoptotic DNA through TUNEL analysis. The assessment of apoptosis by TUNEL staining revealed that there were more apoptotic cells in combinational therapy tissues than controls (Fig. 5B).

image

Figure 5.  Pathological analysis and apoptosis detection in tumor tissues. (A) Fixed sections of tumors were stained with HE. The surface area of necrotic tissue relative to total surface area of the field of view was quantified by morphometric analysis. (B) Apoptosis in xenograft tumors was determined by TUNEL analysis. *< 0.05.

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Decreased angiogenesis and phosphorylation of Akt in tumors.  Immunohistochemical staining of anti-CD31 in tumor tissue from LY294002, sVEGFR2 and LY294002 + sVEGFR2 treatment mice showed significantly decreased microvessel density compared with the control group. The anti-angiogenesis effect induced by LY294002 + sVEGFR2 was stronger than LY294002 and sVEGFR2 alone (Fig. 6A). Immunohistochemical staining of phosphorylated Akt showed that the activation of the PI3K/Akt pathway increased in the sVEGFR2 group might result from the stimulation of hypoxia and low serum because of the decrease of angiogenesis, while significantly decreased in tumors that received treatments with LY294002 or LY294002 + sVEGFR2 (Fig. 6B). Quantitative analysis showed that the least microvessel density and phosphorylated-Akt ratio simultaneously presented in the combination-therapy group (P < 0.05; Fig. 6). These data clearly indicate that LY294002 effectively blocked activation of the PI3K/Akt pathway during anti-angiogenesis therapy.

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Figure 6.  Immunohistochemical analysis of angiogenesis and phosphorylated Akt in tumors. Angiogenesis (A) and phosphorylation of Akt (B) were evaluated by CD31 and Akt-S473 antibody staining, respectively. The microvessel density (A) and levels of phosphorylation of Akt (B) were quantified by counting the number of microvessels and the percentage of phosphorylated Akt, respectively, from five high-power fields. The proportion of phosphorylated Akt in PBS was defined as 100%. *< 0.05.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

Inhibiting angiogenesis is an effective approach to suppressing tumor growth through cutting off serum and oxygen that are indispensable to tumor growth and metastases.(10) However, some studies have indicated that absence of serum and oxygen could induce over-activation of numerous signal pathways, which would confer tumor cells many traits resistant to apoptosis, hypoxia and nutrient deficiency.(14,15,23,24) Among these signal pathways, the PI3K/Akt pathway is one of the most important ones to cope with the absence of serum and oxygen in the tumor microenvironment.(14,15) Accordingly, significant overactivation of some products in this pathway such as PI3K and phosphorylated Akt has been found frequently in various tumors.(32) In the present study, we have also found that the expression of phosphorylated Akt increased gradually in a modeled low-nutrient and low-oxygen microenvironment in vitro in a time-dependent manner. Similarly, the phosphorylation of Akt was significantly increased in tumor tissues after anti-angiogenic therapy. These results further suggest that anti-angiogenesis therapy or deprivation of serum and oxygen could activate the PI3K/Akt pathway.(14,15,24)

Activation of the PI3K/Akt pathway in tumors can not only inhibit cell apoptosis and prolong cell survival, but also promote cell proliferation through various mechanisms, especially in the absence of serum and oxygen.(20,32,33) In contrast, blocking the PI3K/Akt pathway with antibodies or small-molecule inhibitors can effectively inhibit proliferation and increase apoptosis.(18) LY294002, a specific inhibitor of PI3K, has been widely used to block the PI3K/Akt pathway in tumors for many years.(32) Moreover, it has a sensitized effect on chemotherapy, radiotherapy and targeting therapies of EGFR and IGF-IR.(22,27,28) Also, it still sensitizes A549 cells in hypoxia and PANC-1 (pancreas cancer) cells under nutrient starvation conditions.(15,24) In the present study, we have assessed the effects of LY294002 on various tumor cells (CT26, LL/2 and B16 cells) by treating these cells in various modeled tumor microenvironments in vitro. The results indicate that blockage of the PI3K/Akt pathway could not only inhibit the proliferation of CT26 cells in a dose-dependent manner, but also enhance the inhibition when combined with low-nutrient and/or low-oxygen treatment. The enhancement was closely associated with absent levels of serum and/or oxygen, which were consistent with previous studies.(25) Moreover, our data also indicated that blocking the PI3K/Akt pathway could reverse the growth promotion produced by low serum stimulation, which may be a result from the production of growth factor-like cytokines (such as IL-8)(34) being blocked by inactivating the PI3K/Akt pathway. These findings suggest that this combination obviously had an intensive role in suppression of cell growth and confirmed that inhibiting PI3K could enhance the sensitivity of tumor cells to the absence of serum and oxygen.(15,24)

The activated PI3K/Akt pathway has also been implicated in angiogenesis through inducing the expression of HIF-1α and VEGF, activating endothelial nitric oxide synthase (eNOS) to induce the migration of endothelial cells, increasing the expression of matrix metalloproteinase (MMP) and forming vasculogenic mimicry.(8,18,20,21,35–37) The activation of PI3K/AKT pathway could induce tumor cells migrating and forming vascular-like tubular networks, which could mimic the functions of microvessels to supply oxygen and nutrients to tumors, so the activation of PI3K/AKT pathway would weaken the efficacy of anti-angiogenic therapy in vivo.(8,21,37–38) Accordingly, to get better efficacy from anti-angiogenic therapy, it should be combined with treatment against tumor cells, that is, simultaneously inhibiting the growth of endothelial cells and tumor cells. This concept was first confirmed in the present study in vitro, as described above. In order to observe the efficacy of combination treatment in vivo, we constructed the eukaryotic plasmid of soluble VEGFR2 and then combined it with LY294002 to treat the CT26-bearing mice. The data showed that the inhibition of tumors in the combination-therapy group was significantly higher than single treatments. Immunohistochemical analysis showed that angiogenesis and phosphorylated Akt were simultaneously decreased in combined-therapy tumors. Consequently, more apoptotic tumor cells were detected in combinational treatment tissues than any single treatment ones. The findings in vivo further suggested that inhibiting PI3K with LY294002 in tumors enhanced the efficacy of anti-angiogenic therapy. The mechanisms of enhancement might be associated with inhibition of the activated PI3K/Akt pathway during anti-angiogenic therapy. On one hand, anti-angiogenic therapy with sVEGFR2 decreases the supplies of nutrients and oxygen and results in inhibition of tumor growth.(3,4) On the other hand, inhibiting PI3K abolishes protection of tumor cells resistant to apoptosis and simultaneously again reduces angiogenesis to some extent.(17,20) Therefore, this combination not only inhibits the proliferation of endothelial cells for angiogenesis, but also decreases the apoptotic threshold of tumor cells.

Taken together, the combination of blocking activation of the PI3K/Akt pathway with anti-angiogenesis therapy is an effective approach to enhance the antitumor efficacy by simultaneously acting on tumor cells and tumor-associated blood vessel endothelial cells. Therefore, the present study might provide a new therapeutic strategy for cancers resistant to anti-angiogenesis therapy.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

This work was supported by the National 973 Basic Research Program in China (2010CB529900).

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgment
  7. Disclosure Statement
  8. References
  9. Supporting Information

Fig. S1. A MTT assay was used to evaluate the inhibition of LY294002 on CT26 cells in normal conditions.

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CAS_1979_sm_fS1.doc58KSupporting info item

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