SEARCH

SEARCH BY CITATION

Abstract

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

We previously reported that 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) effectively suppresses the growth of solid tumors, likely via its anti-angiogenic activity. To investigate how SQMG affects angiogenesis, we performed DNA microarray analysis and quantitative real-time polymerase chain reaction. Consequently, upregulation of thrombospondin 1 (TSP-1) in SQMG-treated tumors in vitro and in vivo was confirmed. To address the mechanisms of TSP-1 upregulation by SQMG, we established stable TSP-1-knockdown transformants (TSP1-KT) by short hairpin RNA induction and performed reporter assay and in vivo assessment of anti-tumor assay. On the reporter assay, transcriptional upregulation of TSP-1 in TSP1-KT could not be induced by SQMG, thus suggesting that TSP-1 upregulation by SQMG occurred via TSP-1 molecule. In addition, growth of TSP1-KT xenografted tumors in vivo was not inhibited by SQMG, thus suggesting that anti-angiogenesis via TSP-1 upregulation induced by SQMG did not occur, as the SQMG target molecule TSP-1 was knocked down in TSP1-KT transformants. These data provide that SQMG is a promising candidate for the treatment of tumor-induced angiogenesis via TSP-1 upregulation. (Cancer Sci, doi: 10.1111/j.1349-7006.2012.02333.x, 2012)

Angiogenesis, the formation of new blood vessels, is a fundamental process that is necessary for normal embryonic development and is also involved in the development of pathological conditions such as cancer.[1, 2] Its importance in solid tumor growth and metastasis has been widely recognized by multiple studies.[2] A major pathway in tumor angiogenesis involves the vascular endothelial growth factor (VEGF) family of proteins and receptors.[3, 4] Anti-angiogenic treatments are promising therapies for the treatment of cancer: for example, agents such as anti-VEGF antibodies that inhibit VEGF receptor tyrosine kinase and result in effective inhibition of solid tumor growth in vivo have been reported.[5, 6]

Endogenous angiogenesis through VEGF/VEGF-receptor signaling is regulated by thrombospondin 1 (TSP-1). Expression of TSP-1 leads to the inhibition of angiogenic responses such as endothelial proliferation.[7-9] In addition, TSP-1 is involved in several other anti-angiogenic responses, such as inhibition of VEGF-stimulated VEGF receptor-2 (VEGFR-2) signaling[10, 11] and VEGF trapping by its heparin sulfate proteoglycan domain.[12]

We previously reported that the growth of human adenocarcinoma tumors treated with 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) was inhibited, and that these tumors showed extensive hemorrhagic necrosis on histopathological examination.[13, 14] We also confirmed that only some tumor cell lines were sensitive to SQMG, and that angiogenesis was reduced in vivo in SQMG-sensitive tumors, but not in SQMG-resistant tumors.[15] Moreover, although a significant difference in VEGF gene expression was not detected in either SQMG-sensitive or SQMG-resistant tumors, significant downregulation of Tie2 gene expression was observed in all of SQMG-sensitive tumors when compared with controls, but this downregulation was not observed in SQMG-resistant tumors.[15] These data suggest that the anti-tumor effects of SQMG can be attributed to the inhibition of angiogenesis.

Regardless of whether tumors are SQMG sensitive or resistant, the induced blood vessels are considered to be biologically similar. However, differences in the amount of Tie2 gene expression in the endothelial cells are observed. Therefore, further investigation is necessary to understand the mechanisms underlying the anti-angiogenic effects of SQMG. In this study, we confirmed that SQMG treatment in vitro and in vivo results in the upregulation of the TSP-1 gene in SQMG-sensitive tumors, but not in SQMG-resistant tumors, and that the effects of SQMG are nullified in TSP-1 knockdown-sensitive tumors, thus suggesting that the target for SQMG is TSP-1 gene expression.

Materials and Methods

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

Synthesis of SQMG

The chemical structure of the synthesized compound 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) containing fatty acid 18:1 (oleic acid: C18:1) is shown in Figure 1(a). The procedure for the synthesis of SQMG was described previously.[14]

image

Figure 1. 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) structure and in vivo assessment of SQMG for cDNA microarrays study. (a) Structure of SQMG. 3′-sulfoquinovosyl-1′-monoacylglycerol contains a single fatty acid, R = C18:1. 106 tumor cells of MDA-MB-231 (b) or TE-8 (c) were subcutaneously injected into mice. When the solid tumors grew to 30–40 mm3 in tumor volume, mice were injected with SQMG at a dose of 20 mg/kg (SQMG 20) or saline (control) daily for 14 days. Tumor volume from each mouse is shown.

Download figure to PowerPoint

Cell lines

Human breast adenocarcinoma MDA-MB-231 was provided by the Japanese Cancer Research Resources Bank. The cells were cultured with DMEM supplemented with 10% FCS, 100 unit/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine. Human esophagus squamous cell carcinoma TE-8 was obtained from the Health Science Research Resources Bank (Sendai, Japan). These cells were cultured with RPMI 1640 supplemented with 10% FCS, 100 unit/mL penicillin, 100 μg/mL streptomycin and 2 mM l-glutamine.

Establishment of stable transfectants

Total RNA derived from human umbilical vein endothelial cells (HUVEC) (Cambrex, Walkerville, MD, USA) was converted to cDNA by a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science, Mannheim, Germany), which was used to obtain human TSP-1 cDNA by PCR amplification with the following primer sets: forward primer, 5′-GCACCAACAGCTCCACCATG-3′ and reverse primer, 5′-GGGATCTCTACATTCGTATT-3′. The amplified fragment was cloned using the pCR4-TOPO cloning vector (Invitrogen, Carlsbad, CA, USA), and reconstructed with pcDNA3.1 (Invitrogen) at the HindIII/XhoI site. TE-8 cells were transfected with TSP-1 cDNA in the pcDNA3.1 vector, and selected and cloned in a 0.5 mg/mL G418-containing medium (Invitrogen). Several clones were established (TSP1-OT).

We designed five shRNAs and used the MissionRNAi (Sigma-Aldrich, St. Louis, MO, USA) technology platform to stably knockdown TSP-1 gene expression in MDA-MB-231 cells. These sequences were as follows: sh-1, 5′-CCGGATCATCTGGTATACCATTGCCCTCGAGGGCAATGGTATACCAGATGATTTTTTG-3′, sh-2, 5′-CCGGGCGTTGGTGATGTAACAGAAACTCGAGTTTCTGTTACATCACCAACGCTTTTTG-3′, sh-3, 5′-CC-GGCGATGACATCTGTCCTGAGAACTCGAGTTCTCAGGA-CAGATGTCATCGTTTTTG-3′, sh-4, 5′-CCGGCCTTGACAACAACGTGGTGAACTCGAGTTCACCACGTTGTTGTCA-AGGTTTTTG-3′, sh-5, 5′-CCGGCTCTCAAGAAATGGTGTTCTTCTCGAGAAGAACACCATTTCTTGAGAGTTTTTG-3′. The five shRNAs were cloned into the pLKO.1-puro shRNA vector. Plasmid DNA, including non-targeting shRNA as a control (sh-control), was transfected into the MDA-MB-231 cells along with Lentiviral Packaging Mix (Sigma-Aldrich), which consists of an envelope and packaging vector to produce lentivirus packed with shRNA cassettes, using the standard procedure. After transfection, cells were cultured and cloned in the presence of 5 μg/mL puromycin.

In vivo assessment of anti-tumor assay

Inbred female BALB/c nu/nu mice (20–22 g, 7 weeks old) were obtained from Japan SLC, Inc. (Shizuoka, Japan). All procedures were performed in compliance with the guidelines of the Animal Research Committee of Azabu University. Cells (106 cells/mouse) suspended in phosphate-buffered saline (PBS) were injected subcutaneously into the dorsal region of the mice. After implantation, the tumor sizes were measured at 2-day intervals in each mouse. When the solid tumors grew to 30–40 mm3 in tumor volume (tumor volume = length × [width]2 × 0.5), SQMG was administrated daily for 14 days, and tumor growth was observed. Each type of tumor was divided randomly into two groups (= 1, 3, or 4 per group). Mice in the control group were intraperitoneally injected with 0.2 mL of saline solution, and mice in the test groups were intraperitoneally injected with SQMG at a dose of 20 mg/kg daily for 14 days. On the day after the last administration of SQMG, the tumor size was measured, and the tumors were excised and prepared for further study. The mean ± SE of the tumor volume of each group (n = 4 per group) was calculated. The growth of each tumor was analyzed using Student's t-test.

Immunohistochemical study

All of the tumors excised from the mice (n = 4 per group) were embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek USA, Torrance, CA, USA) and frozen. Acetone-fixed cryosections were stained with an anti-mouse CD31 mAb, followed by anti-rat IgG conjugated with AlexaFlour 488 (BD Bioscience Pharmingen, San Jose, CA, USA) as a secondary antibody. Nuclei were counterstained with propidium iodide (PI) (Vector Laboratories, Burlingame, CA, USA). The CD31-positive/ring-form blood vessels in 500-mm2 section areas of these samples were counted at 100× magnification under a fluorescence microscope (Olympus AX80, Olympus, Tokyo, Japan). The data are represented as a mean ± SE of four section areas from each group, and the results were analyzed using Student's t-test.

MTT assay

To investigate the cell growth of the transfectants, the MTT assay was performed according to the methods described previously.[14] Briefly, cells (5 × 103 cells per well) were cultured in 96-well plates for 24 h and different amounts of SQMG suspended in PBS were then added to the wells. After cultivation for 48 h, 50 μg of MTT was added to the cells and incubation was continued for 3 h. Next 4% HCl in 2-propanol was added to each well and mixed using a pipette to disrupt the cells. The absorbance of the contents in each well was measured using a multiwell scanning photometer (Micro ELISA MR600; Dynatech Laboratories, Alexandria, VA, USA) at a wavelength of 570 nm. Results are represented as a mean ± SE of triplicate wells for one of three independent experiments.

Gene expression profiling using cDNA microarrays

We isolated total RNA from the xenografted solid tumors in mice in accordance with protocols recommended by Affymetrix (Santa Clara, CA, USA) for GeneChip experiments. Total RNA was isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, reverse-transcribed to cDNA with a Transcriptor First Strand cDNA Synthesis Kit (Roche Applied Science), and then labeled with a biotinylated nucleotide analog (pseudouridine base). The labeled cDNA products were fragmented, loaded on to the Human Genome U133 Plus 2.0 Array (Affymetrix) and hybridized according to the manufacturer's protocol. The GeneChip array data were analyzed using the DNA Microarray Viewer, provided by Kurabo Industries (Osaka, Japan), which is the authorized service provider of Affymetrix in Japan.

Quantitative real-time RT-PCR analysis

The quantitative measurement of gene expression was performed using the LightCycler system (Roche Applied Science). Quantitative real-time RT-PCR analyses of human glucose-6-phosphate dehydrogenase (G6PDH) and TSP-1 gene expression were performed using the LightCycler FastStart DNA MasterPLUS SYBR Green I system (Roche Applied Science) with the following primer sets: forward primer, 5′-CTGCGTTATCCTCACCTTC-3′ and reverse primer, 5′-CGGACGTCATCTGAGTTG-3′ for the detection of human G6PDH; forward primer, 5′-GATGGAGAATGCTGAGTTG-3′and reverse primer, 5′-TGAGGAGGACACTGGTAGAG-3′ for the detection of human TSP-1. PCR amplification of the housekeeping gene, G6PDH, was performed for each sample as a control for sample loading and to allow for normalization among the samples. To determine the absolute copy number of the target transcripts, the fragments of G6PDH or the target genes amplified by PCR using the above described primer sets were subcloned into the pCR4-TOPO- cloning vector (Invitrogen). The concentrations of these purified plasmids were measured by absorbance at 260 nm, and copy numbers were calculated from the concentration of the samples. A standard curve was created by plotting the threshold cycle (Ct) versus the known copy number for each plasmid template in the dilutions. The copy numbers for all unknown samples were determined according to the standard curve using LightCycler version 3.5.3 (Roche Applied Science). To correct for differences in both RNA quality and quantity between samples, each target gene was first normalized by dividing the copy number of the target by the copy number of G6PDH; therefore, the mRNA copy number of the target was the copy number per the copy number of G6PDH. The initial value was also corrected for the amount of G6PDH indicated as 100% to evaluate the sequential alteration of the mRNA expression level.

Assessment of TSP-1 expression in vitro

Cells were seeded into six-well plates at a density of 2 × 105 cells/well and cultured overnight. Then cells were cultured in the presence of 25 μM SQMG for 0, 1, 3, 6, 12, or 24 h and then harvested. Total RNAs from these cells were prepared and TSP-1 gene expression were performed using same methods for xenografted solid tumors in vivo.

TSP-1 promoter-reporter constructs and luciferase assay

Human genomes were prepared from MDA-MB-231 cells using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer's instructions. A luciferase reporter plasmid containing the −2220 to +750 region of the human TSP-1 gene promoter was cloned from human genome by PCR system using forward primer, 5′-CAACTGAAGTATCATGATAAGAG-3′ and reverse primer 5′-ATCCTGTAGCAGGAAGCACAAG-3′. To ligate into pGL4.10 vector (Promega, Madison, WI, USA), second PCR as template for first PCR product was performed using forward primer; 5′-GTACCGGTACCCAACTGAAGTATCATGATAAGAG-3′, which was added to 5′-GTACCGGTACC-3′ containing KpnI site and reverse primer; 5′-ATATCCTCGAGATCCTGTAGCAGGAAGCACAAG-3′, which was added to 5′-ATATCCTCGAG-3′ containing XhoI site. The PCRs were performed using GoTaq system (Promega). The second PCR product was digested with KpnI and XhoI and subcloned into a luciferase plasmid pGL 4.10 vector (TSP1-promoter pGL4.1 plasmid), and their sequences were confirmed by sequencing.

Thrombospondin 1-knocked down MDA-MB-231 cells (TSP1-KD) or sh-control cells were seeded into 24-well plates at a density of 5 × 104 cells/well and cultured overnight. Then cells were transiently cotransfected using Lipofectamine LTX (Invitrogen) with 40 ng pGL4.74 plasmid (hRluc/TK; Promega) as an internal control, and 1 μg target plasmids, such as SV40 promoter pGL 4.13 plasmid (luc2/SV40; Promega) as a positive control, the promoterless pGL 4.10 plasmid as negative vector, or TSP1-promoter pGL 4.10 plasmid. After 24 h of transfection, these cells were cultured with 25 μM SQMG or not for 1, 3, 6, 12 or 24 h. Luciferase activities were assayed using the dual-luciferase assay kit (Promega) according to the manufacturer's directions. The relative luciferase activity for each cell was calculated relative to the activity of the promoterless pGL 4.10 plasmid (negative control). The results were analyzed using Student's t-test.

Results

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

Gene expression profiling using cDNA microarrays

3′-sulfoquinovosyl-1′-monoacylglycerol-sensitive MDA-MB-231 cells and SQMG-resistant TE-8 cells were subcutaneously injected into mice. Mice bearing solid tumors that grew to 30–40 mm3 were then intraperitoneally injected with saline or SQMG daily for 14 days. As shown in Figure 1(b), the SQMG treatment in mouse with MDA-MB-231 solid tumor resulted in inhibition of tumor growth as compared with control mouse on the day after the last injection. In contrast, mouse with TE-8 solid tumor treated with SQMG showed no inhibition of tumor growth as compared with control mouse on the day after the last injection (Fig. 1c).

Four solid tumors were harvested, total RNA was prepared and gene expression profiles were analyzed using cDNA microarrays. A subset of the microarray results is shown in Table 1. Genes that were upregulated after SQMG treatment in the SQMG-sensitive tumor MDA-MB-231 samples, as compared with controls, and that also showed no change in expression in the TE-8 sample, regardless of SQMG administration, were identified. Given its known anti-angiogenic effects, we chose to investigate TSP-1 as a potential target of SQMG.[7-9] In order to confirm the upregulation of TSP-1 gene expression in xenografted tumors in nude mice, we again performed in vivo assessment and real-time PCR. Total RNAs derived from four SQMG-sensitive MDA-MB-231 tumors treated with SQMG and three SQMG-sensitive tumors treated without SQMG, and from a total of eight SQMG-resistant TE-8 tumors treated either with or without SQMG, were also harvested. As shown in Figure 2(a), in the SQMG-sensitive MDA-MB-231 tumors, an increased tendency of TSP-1 expression with SQMG treatment was observed (= 0.11). However, in the SQMG-resistant TE-8 tumors, a difference in expression was not observed (Fig. 2b). Therefore, we further investigated whether SQMG treatment could induce upregulation of TSP-1 gene expression in MDA-MB-231 and TE-8 cells in vitro. Consequently, an increase in TSP-1 gene expression in MDA-MB-231 was observed from 3 to 12 h after SQMG treatment, and this returned to baseline by 24 h, indicating that the duration of upregulation is short (Fig. 2c). On the other hand, upregulation in TE-8 was not observed. These data suggest that the upregulation of TSP-1 gene expression is induced by SQMG in SQMG-sensitive MDA-MB-231 cells in vitro and that detection in xenografted solid tumors is difficult in vivo because the duration of upregulation is short.

image

Figure 2. Upregulation of thrombospondin 1 (TSP-1) gene expression by 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) in MDA-MB-231 cells. TSP-1 mRNA copy number in xenografted tumors derived from MDA-MB-231 (a) or TE-8 (b) treated with or without SQMG in vivo were measured by quantitative real time polymerase chain reaction (PCR). Data are presented as TSP-1 mRNA copy number per the copy number of G6PDH indicating by open circles. Bar indicates means of each test group. (c) The means ± standard error (SE) of TSP-1 mRNA copy numbers of MDA-MB-231 (dark gray bar) or TE-8 (light gray bar) treated with 25 μM SQMG in vitro are presented, which normalized with the mRNA copy number of G6PDH.

Download figure to PowerPoint

Table 1. Results of DNA micro array analysis (gene expression SQMG treatment/control)
 SQMG/control
MDA-MB-231TE-8
ATP-binding cassette, sub-family F, member 24.31
E2F transcription factor 74.30.9
Inhibitor of DNA binding 2, dominant negative helix-loop-helix3.70.9
Lysyl oxidase30.9
Gap junction protein, alpha 1, 43 kDa2.81
Egl nine homolog 3 (C. elegans)2.80.9
Angiogenin2.60.9
v-myc myelocytomatosis viral oncogene homolog (avian)2.51
Thrombospondin-11.70.9
Serpin peptidase inhibitor, clade B (ovalbumin), member 50.10.9
Vitamin D (1,25- dihydroxyvitamin D3) receptor0.21

TSP-1-overexpressing transformants were unable to grow in vivo

In order to confirm the anti-angiogenic effects of TSP-1, SQMG-resistant TE-8 cells were transfected with TSP-1 cDNA and TSP-1-overexpressing transformants (TSP1-OT) were cloned. We selected three clones (i.e., clones 4, 8 and 13) that showed approximately threefold expression of TSP-1, as compared with intact TE-8 or empty vector (pcDNA3.1) control (Fig. 3a). These three TSP1-OT clones had similar growth rates in vitro when compared with controls (Fig. 3b). However, the TSP1-OT clone cells (TSP1-OT clone 4) did not grow in vivo (Fig. 3c). Implantation using other clones, such as clones 8 and 13, was also attempted; however, growth of these clones in vivo was not observed. These data suggest that overexpression of TSP-1 is critical for tumorigenesis.

image

Figure 3. Establishment of thrombospondin 1 (TSP-1)-overexpressed transformant (TSP1-OT) and the assessment of tumorigenesis in vivo. TE-8 cells were transfected with TSP-1 cDNA in pcDNA3.1 vector or empty vector, and selected, cloned, and established several clones (TSP1-OT). (a) TSP-1 mRNA copy number of TSP-1-overexpressed transformants were measured by real time polymerase chain reaction (PCR). (b) Growth rate of three TSP1-OT clones in vitro were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (c) 106TSP1-OT clone 4 or control cells were subcutaneously injected into mice and the tumor sizes were measured at 2-day intervals in each mouse. The means ± standard error (SE) of tumor volumes from each group (= 4/group) are shown.

Download figure to PowerPoint

Establishment and characterization of stable TSP-1-knockdown transformant

We designed five shRNAs and used the Mission RNAi (Sigma-Aldrich) technology platform to stably knockdown TSP-1 gene expression in SQMG-sensitive MDA-MB-231 cells. When sh-2 shRNA was transfected into the cells, the endogenous TSP-1 gene expression decreased by approximately 92%, as compared with the sh-control treated with shRNA for a non-target gene (Fig. 4a). Among the five shRNAs, sh-2 most effectively induced the downregulation of TSP-1 expression. Using the sh-2 transformants, we successfully obtained a stable TSP-1 knockdown transformant clone, clone 80 (TSP1-KT), which showed approximately 94% reduced TSP-1 gene expression when compared with the sh-control (Fig. 4b). We further investigated the growth rate of TSP1-KT (clone 80) in vitro using MTT assay. No differences in growth rates were observed between TSP1-KT and the sh-control was observed, thus suggesting that TSP-1 did not influence in vitro growth activity (Fig. 4c).

image

Figure 4. Establishment of thrombospondin 1 (TSP-1)-knockdown transformant clone (TSP1-KT) and the assessment of tumorigenesis in vivo. (a) Plasmid DNA including five TSP-1-targeting (sh1–5) or non-targeting shRNAs (sh-control) ligated into pLKO.1-puro shRNA vector were transfected into MDA-MB-231 cells along with Lentiviral Packaging Mix. TSP-1 mRNA copy number of these TSP1-KT clones were measured by real time polymerase chain reaction (PCR). (b) MDA-MB-231 cells were introduced sh-2 shRNA, and selected, cloned, and TSP-1 mRNA copy number of these cells was measured by real time PCR. (c) Growth rate of TSP1-KT clone (clone 80) in vitro were measured by by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (d and e) Data of reporter assay using TSP-1 promoter. sh-control (d) and TSP1-KT (e) cells were transfected with reporter plasmids together with pGL4.74 plasmid as an internal control and then cultured with or without 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) representing S or C, respectively. The relative luciferase activity for each cell was calculated relative to the activity of the promoterless plasmid (negative control representing NC). Data are the means ± standard error (SE) (= 3 in three trials; *< 0.05). (f and g) In vivo assessment of anti-tumor effects of SQMG using TSP1-KT. 106TSP1-KT (g) or control cells (f) were subcutaneously injected into mice. When the solid tumors grew to 30–40 mm3 in tumor volume, mice were injected with SQMG at a dose of 20 mg/kg (SQMG 20) or saline (control) daily for 14 days. The means ± SE of tumor volumes from each group (n = 4/group) are shown. *< 0.01.

Download figure to PowerPoint

To investigate the mechanism of TSP-1 upregulation by SQMG, we performed Dual-Luciferase reporter assay. If SQMG stimulation is transduced via TSP-1 molecules to induce TSP-1 expression, TSP1-KT would have no effect. As cDNA encoding TSP-1-targeting sh-RNA is stably expressed in TSP1-KT, we were unable to detect TSP-1 gene expression. Therefore, to investigate this signal transduction, we used a Dual-Luciferase reporter assay using the TSP-1 promoter that would not influence the sh-RNA system.[16] When sh-control cells were introduced with luciferase reporter constructs containing the TSP-1-promoter, luciferase expression of SQMG-treated cells was significantly increased from 1 to 3 h after treatment as compared with non-treatment control (< 0.05, Fig. 4d). These data demonstrate that SQMG is able to induce TSP-1 gene expression, which supports the real-time PCR data shown in Fig. 2c, although a time lag was observed. On the other hand, when TSP1-KT was introduced with luciferase reporter constructs with culture in the presence or absence of SQMG, no differences were observed, thus suggesting that TSP-1 upregulation by SQMG occur via TSP-1 molecule (Fig. 4e).

The tumorigenic potential of TSP1-KT in vivo was then investigated by subcutaneously injecting them into mice, and mice bearing solid tumors that grew to 30–40 mm3 in volume were then injected intraperitoneally with saline or SQMG daily for 14 days (days 16–30). As shown in Figure 4(f), SQMG treatment of mice bearing sh-control solid tumors resulted in significant inhibition of tumor growth when compared with the saline-treated control group (P < 0.05). In contrast, SQMG treatment of mice bearing TSP1-KT solid tumors did not lead to tumor growth inhibition, as compared with the control group (Fig. 4g). These data confirm that growth of TSP1-KT solid tumors is not inhibited by SQMG treatment, thus suggesting that anti-angiogenesis via TSP-1 upregulation induced by SQMG treatment did not occur, as the SQMG target molecule TSP-1 was knocked down in TSP1-KT transformants.

In accordance with our previous study, we further performed immunohistochemical analysis to determine the angiogenesis profiles of tumors in an effort to confirm the absence of anti-tumor effects by SQMG treatment in TSP1-KT tumors.[15] Tumors were excised from the mice and cryosections of the acetone-fixed tumors were stained with anti-mouse CD31 mAb as an endothelial cell marker. Representative photographs showing immunohistochemical staining of tumors treated with or without SQMG are shown in Figure 5. CD31-positive/ring-form blood vessels were clearly observed for both the sh-control (Fig. 5a,b) and TSP1-KT (Fig. 5c,d), and were counted for all samples in 500-mm2 section areas under a fluorescence microscope. As shown in Figure 5(e), the number of blood vessels was significantly higher (P < 0.01) in TSP1-KT control tumors when compared with sh-control control tumors, thus suggesting that the downregulation of TSP-1 facilitated more angiogenesis. In contrast, in TSP1-KT tumors treated with or without SQMG, no significant differences in the number of blood vessels were observed. However, the number of blood vessels in the sh-control tumors was significantly lower (P < 0.05) after SQMG treatment when compared with TSP1-KT tumors.

image

Figure 5. Assessment of antiangiogenesis by immunohistochemical staining. Cryosections of sh-control tumors treated with (a) and without (b) 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG) were stained by anti-mouse CD31 mAb/anti-rat IgG conjugated with AlexaFlour 488, and nuclei were counterstained with PI. TSP1-KT tumors treated with (c) and without (d) SQMG were stained by anti-mouse CD31 mAb/anti-rat IgG conjugated with AlexaFlour 488, and nuclei were counterstained with propidium iodide. Arrows indicate CD31-positive blood vessels. Scale bar = 100 μm.

Download figure to PowerPoint

Discussion

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

In this study, we showed that SQMG is not anti-angiogenic in TSP-1-knockdown xenograft tumors, thus suggesting that the anti-tumor effects of SQMG can be attributed to the inhibition of tumor angiogenesis via the upregulation of TSP-1. TSP-1 is an oligomeric extracelluar matrix glycoprotein that mediates cell–cell and cell–matrix interactions by binding to cell-surface receptors, such as CD36 and integrin-associated protein (IAP)/CD47.[9, 17-19] Several mechanisms have been reported to explain the anti-angiogenic effects of TSP-1 including endothelial cell apoptosis.[7-9, 20] The association between TSP-1 and CD36, which is known to be expressed on endothelial cells and is a receptor for TSP-1, induces endothelial cell apoptosis through the sequential activation of Fyn phosphorylation, caspase-3-like proteases and p38 MAPK activity.[9, 21] Thrombospondin 1 has also been reported to have an effect on VEGF-A-induced phosphorylation of VEGFR2 via the activation of CD36, which induces the inhibition of endothelial migration.[22] Moreover, in the absence of TSP-1, the association of VEGFR-2 with CD36 and spleen tyrosine kinase (Syk) decreased and Syk promoted VEGF-A-induced endothelial cell activity via VEGFR-2 phosphorylation.[23] The association between TSP-1 and CD36 is a major pathway in the inhibition of angiogenesis and ligation of CD47 is reported to inhibit nitric oxide-stimulated angiogenesis and VEGFR2 activation.[18, 24] Thus, TSP-1 possesses anti-angiogenic activity, and its upregulation by SQMG results in significant antitumor activity, although these effects depend on the cellular context.

Whether a tumor is sensitive or resistant to SQMG may depend on whether TSP-1 is upregulated in that tumor. To address the mechanism of how TSP-1 is upregulated by SQMG treatment, we investigated the amount of TSP-1 mRNA by real-time PCR and the transcriptional mechanism on the TSP-1 promoter by luciferase reporter assay in vitro. Although the mechanism of TSP-1-upregulation by SQMG in xenografted solid tumor in vivo has not been fully demonstrated, the presented data suggested that the TSP-1-upregulation by SQMG is necessary for the existence of a signal transduction pathway via TSP-1 molecule. The master regulator of TSP-1 gene expression is known to be p53.[25] Gene expression under the regulation of p53 involves multiple mechanisms, such as post-translational modifications and the availability of transcriptional cofactors that can regulate the activity of p53.[25-27] Indeed, regulation of TSP-1 by p53 appears to vary in different cell and tissue types: for example, TSP-1 expression is correlated with p53 status in prostate cancer,[28, 29] liver,[30] ovarian carcinoma[31] and glioma,[32] but is inversely correlated with p53 status in colon cancer.[33] These data suggest that the regulation of TSP-1 expression by p53 is cell context-dependent and that the expression of different p53-binding cofactors may modulate this regulation. In addition to several transcriptional cofactors, post-transcriptional microRNA (miRNA)-based mechanisms involved in the regulation of TSP-1 were also recently reported. cMyc-activated miR-17-92 was shown to be involved in the downregulation of TSP-1,[34] and p53-activated miR-194 was found to inhibit TSP-1 and promote angiogenesis in colon cancer.[35] This study did not elucidate whether the difference in sensitivity to SQMG was correlated with the cell context-dependent regulation of TSP-1 expression by p53.

We previously reported that significant downregulation of Tie2 gene expression in endothelial/pericyte cells was observed in all SQMG-sensitive tumors when compared with controls.[16] Results from this study suggest that the anti-tumor effects of SQMG are attributed to the inhibition of tumor angiogenesis via upregulation of TSP-1. It is assumed that TSP-1 upregulation in tumor cells causes Tie2 downregulation in endothelial/pericyte cells, given that SQMG is not anti-angiogenic in the TSP-1 knockdown xenograft tumors. Although little is known about TSP-1-inducible chemical compounds, such a trichostatin A, SQMG is a promising candidate for the treatment of tumor-induced angiogenesis via TSP-1 upregulation.[36]

Acknowledgments

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

This research was supported by The Special Coordination Funds on Science and Technology of the Ministry of Education, Culture, Sports, Science of Japan and a research project grant awarded by the Azabu University.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  • 1
    Folkman J. What is the evidence that tumor are angiogenesis dependent? J Natl Cancer Inst 1990; 82: 46.
  • 2
    Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005; 438: 9326.
  • 3
    Hicklin DJ, Ellis LM. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. J Clin Oncol 2005; 23: 101127.
  • 4
    Carmeliet P, Jain RK. Molecular mechanisms and clinical applications of angiogenesis. Nature 2011; 473: 298307.
  • 5
    Hurwitz H, Feherenbacher L, Novotny W et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med 2004; 350: 233542.
  • 6
    Ferrara N, Kerbel RS. Angiogenesis as a therapeutic target. Nature 2005; 438: 96774.
  • 7
    Bagvandoss P, Wilks JW. Specific inhibition of endothelial cell proliferation by thrombospondin. Biochem Biophys Res Commun 1990; 170: 86772.
  • 8
    Good DJ, Polverini PJ, Rastinejad F et al. A tumor suppressor-dependent inhibitor of angiogenesis is immunologically functionally indistinguishable from a fragment of thrombospondin. Proc Natl Acad Sci USA 1990; 87: 66248.
  • 9
    Dawson DW, Pearce SF, Zhong R, Silverstein RL, Frazier WA, Bouck NP. CD36 mediates the in vitro inhibitory effects of thrombospondin-1 on endothelial cells. J Cell Biol 1997; 138: 70717.
  • 10
    Zang X, Kazerounian S, Duquette M et al. Thrombospondin-1 modulated vascular endothelial growth factor activity at the receptor level. FASEB J 2009; 23: 336876.
  • 11
    Kaur S, Martin-Manso G, Pendrak ML, Garfield SH, Isenberg JS, Robert DD. Thrombospondin-1 inhibit VEGF receptor-2 signaling by disrupting its association with CD47. J Biol Chem 2010; 285: 3892332.
  • 12
    Gupta K, Gupta P, Wild R, Ramakrishnan S, Hebbel RP. Binding and displacement of vascular endothelial growth factor (VEGF) by thrombospondin: effect on human microvascular endothelial cell proliferation and angiogenesis. Angiogenesis 1999; 3: 14758.
  • 13
    Sahara H, Ishikawa M, Takahashi N et al. In vivo anti-tumour effect of 3′-sulphonoquinovosyl 1′-monoacylglyceride isolated from sea urchin (Strongylocentrotus intermedius) intestine. Br J Cancer 1997; 75: 32432.
  • 14
    Sahara H, Hanashima S, Yamazaki T et al. Anti-tumor effect of chemically synthesized sulfolipids based on sea urchin's natural sulfonoquinovosylmonoacylglycerols. Jpn J Cancer Res 2002; 93: 8592.
  • 15
    Mori Y, Sahara H, Matsumoto K et al. Downregulation of Tie2 gene by a novel antitumor sulfolipid, 3′-sulfoquinovosyl-1′-monoacylglycerol (SQMG), targeting angiogenesis. Cancer Sci 2008; 99: 106370.
  • 16
    Donoviel DB, Framson P, Eldrige CF, Cooke M, Kobayashi S, Bornstein P. Structural analysis and expression of the human thrombospondin gene promoter. J Biol Chem 1988; 263: 185903.
  • 17
    Isenberg JS, Jia Y, Fukuyama J Switzer CH, Wink DA, Roberts DD. Thrombospondin-1 inhibits nitric oxide signaling via CD36 by inhibiting myristic acid uptake. J Biol Chem 2007; 282: 1540415.
  • 18
    Isenberg JS, Ridnour LA, Dimitry J et al. CD47 is necessary for inhibition of nitric oxide-stimulated vascular cell responses by thrombospondin-1. J Biol Chem 2006; 281: 2606980.
  • 19
    Krutzsch HC, Choe BJ, Sipes JM, Guo N, Roberts DD. Identification of an alpha(3)beta(1) integrin recognition sequence in thrombospondin-1. J Biol Chem 1999; 274: 240806.
  • 20
    Isenberg JS, Martin-Manson G, Maxhimer JB, Roberts DD. Regulation of nitric oxide signalling by thrombospondin 1: implications for anti-angiogenic therapies. Nat Rev Cancer 2009; 9: 18294.
  • 21
    Jiménez B, Volpert OV, Crawford SE, Febbraio M, Silverstein RL, Bouck N. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1. Nat Med 2000; 6: 418.
  • 22
    Primo L, Ferrandi C, Roca C et al. Identification of CD36 molecular features required for its in vitro angiostatic activity. FASEB J 2005; 19: 17135.
  • 23
    Kazerounian S, Duquette M, Reyes MA et al. Priming of the vascular endothelial growth factor signaling pathway by thrombospondin-1, CD36, and spleen tyrosine kinase. Blood 2011; 117: 465866.
  • 24
    Kaur S, Martin-Manso G, Pendrak ML, Garfield SH, Isenberg JS, Roberts DD. Thrombospondin-1 inhibits VEGF receptor-2 signaling by disrupting its association with CD47. J Biol Chem 2010; 285: 3892332.
  • 25
    Dameron KM, Volpert OV, Tainsky MA, Bouck N. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 1994; 265: 15824.
  • 26
    Shinobu N, Maeda T, Aso T et al. Physical interaction and functional antagonism between the RNA polymerase II elongation factor ELL and p53. J Biol Chem 1999; 274: 1700310.
  • 27
    Roe JS, Kim H, Lee SM, Kim ST, Cho EJ, Youn HD. p53 stabilization and transactivation by a von Hippel-Lindau protein. Mol Cell 2006; 22: 395405.
  • 28
    Xiao W, Zhang Q, Jiang F, Pins M, Kozlowski JM, Wang Z. Suppression of prostate tumor growth by U19, a novel testosterone-regulated apoptosis inducer. Cancer Res 2003; 63: 4698704.
  • 29
    Kwak C, Jin RJ, Lee C, Park MS, Lee SE. Thrombospondin-1, vascular endothelial growth factor expression and their relationship with p53 status in prostate cancer and benign prostatic hyperplasia. BJU Int 2002; 89: 3039.
  • 30
    Su F, Pascal LE, Xiao W, Wang Z. Tumor suppressor U19/EAF2 regulates thrombospondin-1 expression via p53. Oncogene 2010; 29: 42131.
  • 31
    Alvarez AA, Axelrod JR, Whitaker RS et al. Thrombospondin-1 expression in epithelial ovarian carcinoma: association with p53 status, tumor angiogenesis, and survival in platinum-treated patients. Gynecol Oncol 2001; 82: 2738.
  • 32
    Harada H, Nakagawa K, Saito M et al. Introduction of wild-type p53 enhances thrombospondin-1 expression in human glioma cells. Cancer Lett 2003; 191: 10919.
  • 33
    Tokunaga T, Nakamura M, Oshika Y et al. Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 gene expression in colon cancer cells. Virchows Arch 1998; 433: 4158.
  • 34
    Dews M, Homayouni A, Yu D et al. Augmentation of tumor angiogenesis by a myc-activated microRNA cluster. Nat Genet 2006; 38: 10605.
  • 35
    Sundaram P, Hultine S, Smith LM et al. p53-responsive miR-194 inhibits thrombospondin-1 and promotes angiogenesis in colon cancers. Cancer Res 2011; 71: 7490501.
  • 36
    Kang JH, Kim SA, Chang SY, Hong S, Hong KJ. Inhibition of trichostatin A-induced antiangiogenesis by small-interfering RNA for thrombospondin-1. Exp Mol Med 2007; 39: 40211.