International Journal of Urology (2015) 22, 590--595
doi: 10.1111/iju.12753
Original Article: Laboratory Investigation
Sulfoquinovosylacylpropanediol is a novel potent radiosensitizer in prostate cancer Yugo Sawada,1 Kazuya Omoto,1 Naoki Kohei,1 Kengo Sakaguchi,2 Masahiko Miura3 and Kazunari Tanabe1 1
Department of Urology, Tokyo Women’s Medical University, Tokyo, 2Department of Applied Biology, Faculty of Science and Technology, Tokyo University of Science, Chiba and 3Oral Radiation Oncology, Department of Oral Restitution, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan Abbreviations & Acronyms α-SQMG = α-sulfoquinovosylmonoacylglycerol DAPI = 4´6´-diamindino-2phenylindole dihydrochloride PBS = phosphate-buffered saline PCa = prostate cancer SMA = smooth muscle actin SQAP = sulfoquinovosylacylpropanediol XRT = X-ray irradiation Correspondence: Kazunari Tanabe M.D., Ph.D., Department of Urology, Tokyo Women’s Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo 162-8666, Japan. Email: [email protected]. ac.jp Received 22 November 2014; accepted 11 February 2015. Online publication 17 March 2015
Objectives: To examine the effects of combined treatment with sulfoquinovosylacylpropanediol and X-ray irradiation on the remodeling of the prostate cancer microenvironment, including angiogenic and hypoxic characteristics. Methods: Human prostate cancer cells (DU145 and PC3) were implanted subcutaneously into the right hind legs of athymic nude mice. After the tumor volume reached 100–300 mm3, 2 mg/kg/day sulfoquinovosylacylpropanediol was given intravenously from day 0 to day 4, and cells were exposed to 4 Gy X-ray irradiation on days 0 and 3 (for a total of 8 Gy). Tumors were fixed and stained for pathological analyses and immunohistochemical evaluations. To analyze vascular normalization, 60 mg/kg pimonidazole dissolved in saline was injected intraperitoneally. Results: Combined treatment with sulfoquinovosylacylpropanediol plus X-ray irradiation enhanced growth inhibition in DU145 xenografts. The tumor vessel density in DU145 cells significantly decreased after the combined treatment. Staining for smooth muscle actin in vessels was significantly increased. Pimonidazole staining, showing hypoxic lesions, was negative from 72 h, but positive at 6 and 24 h after the first combined treatment. In contrast, no enhancement of the microenvironment in PC3 xenografts was observed with sulfoquinovosylacylpropanediol plus X-ray irradiation. Conclusion: Sulfoquinovosylacylpropanediol could be a novel potent radiosensitizing agent targeting angiogenesis in prostate cancer.
Key words:
oxygenation, prostate cancer, radiation therapy, radiosensitizer, vascular
normalization.
Introduction PCa is the second most common cause of cancer and the sixth leading cause of cancer death in men worldwide, with an estimated 1.1 million new cases and 307 000 deaths in 2012. The American Cancer Society estimated that 233 000 new cases of PCa will be diagnosed, and that 29 480 men will die of PCa in 2014. Besides skin cancer, PCa is the most common cancer and the second leading cause of cancer death in the USA. Interestingly, the incidence of PCa varies more than 25-fold among different countries worldwide. In Asian countries including Japan, the incidence of PCa continues to increase.1 Although the majority of patients with PCa can be successfully treated with surgery or radiation therapy, recurrence is observed in approximately 20–40% of patients within 10 years of treatment.2 The risk of recurrence is elevated to approximately 50% for patients with locally advanced PCa, a condition that is primarily treated by radiation therapy.3 At this stage in the disease, PCa cells are only moderately responsive or even unresponsive to the cytotoxic effects of chemotherapy or radiotherapy. Increased concentrations of high doses of radiation and cytotoxic agents fail to improve the response to therapy, conferring resistance to apoptosis in PCa cells. Therefore, it is essential to identify anticancer agents that are highly effective, non-toxic to normal cells and induce cell death preferentially in tumor cells. Inhibiting angiogenesis is a promising approach for the treatment of cancer, and various antiangiogenic agents have been developed.4 However, many anti-angiogenic agents are associated with adverse events, such as hemorrhage, thrombotic disease and hypertension.5 Importantly, monotherapy with anti-angiogenic agents is not expected to be an effective cure for cancer. Therefore, we have attempted to identify an effective cancer-specific treatment by combining
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anti-angiogenic agents and chemotherapeutic agents or irradiation. Previous studies have investigated the combined use of angiogenesis inhibitors and radiosensitizers, including hypoxic cell sensitizers; however, no therapeutic regimens have yet been used as standard treatments.6–9 SQAP is a sulfoglyco-lipid originally derived from sea urchins, and they are also naturally found in higher plants or sea algae (Fig. 1).10–12 Our group has established the complete chemical synthesis of SQAP.13,14 Sahara et al. reported that αSQMG, which is a prototype of SQAP, suppresses the growth of human solid tumors transplanted into nude mice, but shows no or little effect on in vitro cultured tumor cells.13 Furthermore, Sakimoto et al. reported that α-SQMG combined with radiation synergistically suppresses tumor growth at low doses that do not have inhibitory effects on tumor growth when administered alone.15 Ohta et al. reported that α-SQMG enhances the radioresponse of tumor tissue, accompanied by significant induction of vascular normalization, leading to oxygenation during radiation therapy.16 However, the effects of SQAP in PCa are not yet known. Therefore, in the present study, we examined the effects of combined SQAP and XRT in PCa cells.
(mm) × width (mm) × height (mm) × π / 6. This study was approved by the Animal Ethics Committee of the Faculty of Medicine, Tokyo Women’s Medical University.
Treatments Synthesized SQAP was prepared 1 day before experimentation and dissolved in saline at the appropriate concentrations. To analyze the tumor radioresponse, 2 mg/kg SQAP (dissolved in 100 μL of saline) was given intravenously to the SQAP group and the XRT + SQAP group five times daily from days 0–4. The same amount of saline was injected into mice in the control group and the XRT group. Non-anesthetized mice were irradiated (4 Gy/fraction) on days 0 and 3 with X-ray therapeutic machines (MBR-1520R-3; Hitati, Tokyo, Japan) while shielding the body with lead. Mice were killed, and the tumors were dissected and rapidly frozen on days 10 and 20. To analyze vascular normalization, the same treatments were carried out as for the tumor radioresponse, except that the mice were irradiated and given SQAP once on day 0, and 60 mg/kg pimonidazole dissolved in saline was injected intraperitoneally 30 min before the mice were killed at the indicated times after starting treatment.
Methods
Colony-forming assay
Cell lines and animal studies
Clonogenic cell survival was evaluated by dose–survival curves using colony-forming assays. Cells were irradiated in plastic tubes, plated in 60-mm dishes and incubated at 37 °C. After 7–10 days of incubation, cells were fixed and stained with crystal violet. Colonies containing more than 50 cells were counted. The surviving fraction is determined as follows. We counted the colonies plated in 60-mm dishes in which tumor cells were not irradiated before incubation. The plating efficiency was calculated as the number of colonies divided by the number of plated cells. Therefore, the ratio of the irradiated colonies to the total number of colonies was considered the survival fraction.
DU145 and PC3 human prostate cancer cells were, obtained from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin and 1 mmol/L sodium pyruvate at 37 °C in a 5% CO2 humidified atmosphere. Viable tumor cells (2 × 106) were implanted subcutaneously into the right hind legs of male BALB/c Slc-nude mice (6–8 weeks-of-age). After the tumor volume reached 100–300 mm3, the tumors were treated, and tumor growth was monitored by palpation. The size of the palpable tumors was measured using calipers every 2 days. The tumor volume (V, mm3) was estimated based on the length
Fig. 1 Chemical structure of SQAP. SQAP consists of three parts: glucose, propanediol and stearic acid. © 2015 The Japanese Urological Association
Immunofluorescence staining and image analysis To analyze the tumor radioresponse, consecutive 10-μm-thick tumor cryosections were fixed in 4% paraformaldehyde in PBS at room temperature, incubated in blocking buffer and then probed overnight at 4 °C with primary antibodies targeting endothelial cells (CD34; 1:100; Abcam Japan, Tokyo, Japan). After extensive washing in PBS, the sections were incubated with Alexa 488-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature. To examine vascular normalization, the cryosections were fixed in 4% formaldehyde, incubated in blocking buffer, and then probed with a Cy3-conjugated antibody specific for pericytes (α-SMA; 1:1000; Sigma, St. Louis, MO, USA) overnight at 4 °C. To detect hypoxia, the sections were fixed in icecold acetone for 10 min, and then incubated with an antipimonidazole primary antibody followed by an Alexa 488-conjugated secondary antibody according to the manufacturer’s instructions (Hypoxyprobe-1; Natural Pharmacia International, Burlington, MA, USA). After extensive washing in PBS, all of the immunostained sections were covered with an 591
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antifade agent containing DAPI (Vector Laboratories, Burlingame, CA, USA) and examined with a fluorescence microscope (Axio Observer Z1; Zeiss, Göttingen, Germany). Tumor sections were imaged using a 4× or 10× objective, digitized after background correction and then analyzed using Photoshop CS6 software (Adobe Systems, San Jose, CA, USA). Eight-bit grayscale images from adjacent microscopic fields were acquired and digitally overlaid for multiple stains. To quantify the stained areas, the grayscale images were converted to binary images, and the number of pixels was counted. The normalization index was calculated as the ratio of α-SMA to CD34. For all quantifications, the data were normalized based on the DAPI intensity, which was stained simultaneously with the specific markers.
Statistical analysis Mean values were statistically compared using Student’s t-test or ANOVA. Differences with P-values >0.05 were considered significant.
the cytotoxic activity of SQAP in Hela cells was only approximately 60% that of α-SQMG, which was used in previous experiments (data not shown).
Radiosensitivities of DU145 and PC3 cells differed Because we expected that the differences observed in Figure 2 might be a result of variations in the radiosensitivities of the cell lines, we examined the effects of XRT alone on cell survival using colony-forming assays. PC3 cells showed significantly increased radiosensitivity compared with DU145 cells in vitro (Fig. 3). Therefore, these data suggested that the radiosensitivity of PC3 cells was too strong to observe any synergistic effects of the combined therapy, as shown in Figure 2.
Results Combination of SQAP and radiation enhanced growth inhibition in DU145 xenografts In the present study, we used DU145 and PC3 prostate cancer cells, and established tumors in mice. Tumors derived from DU145 cells showed greater inhibition of tumor growth after combined treatment in vivo (Fig. 2a). However, tumors derived from PC3 cells showed the most growth inhibition in both the XRT group and the XRT + SQAP group, suggesting that the combined treatment had no synergistic effects in this cell line (Fig. 2b). Notably, SQAP alone did not significantly affect tumor growth in either cell line. Regarding the toxicity of SQAP, we confirmed that there were no side-effects in the mice receiving 20 mg/kg/day SQAP. In addition, MMT assays showed that
Fig. 3 Dose–survival curves in DU145 ( ) and PC3 ( ) cells after irradiation. The surviving fractions were determined by colony-forming assays. *P < 0.05.
Fig. 2 Relative tumor volumes over time. Five injections of SQAP (2 mg/kg, i.v.) plus two fractions of X-ray irradiation (4 Gy/fraction) were given. Vehicle ), SQAP ( ; 2 mg/kg/injection), XRT ( ; 4 Gy/ ( ) groups were analyzed. fraction) and XRT + SQAP ( Arrows indicate injections. Each point represents the mean ± SD of five or six tumors. *P < 0.05: XRT versus XRT plus SQAP.
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Synergistic effects of the combined treatment resulted in increased expression of α-SMA in tumor tissues We used dual staining for CD34 and α-SMA, markers of endothelial cells and pericytes, respectively, to determine whether tumor vascular normalization was induced with combined treatment. To this end, we examined the point at which pericytes were recruited around the tumor vessels after treatment as a characteristic marker of vascular normalization.17 The normalization index was determined as the ratio of the areas stained for α-SMA to those stained for CD34. Up to 24 h after treatment, regions stained for CD34 and α-SMA in DU145-derived tumors did not show remarkable changes. However, there was a significant increase in the normalization index in DU145-derived tumors, including an increase in α-SMA staining 72 h after combined treatment (Fig. 4a,b). In contrast, no changes in the vascular normalization index were observed for PC3-derived tumors (Fig. 4c,d).
Vascular normalization resulting from combined treatment caused decreased hypoxic regions in tumor tissues When vascular normalization is induced in tumor tissue, the hypoxic fraction is decreased.17 To analyze the hypoxic regions in DU145-derived tumors, we used pimonidazole, which forms adducts in cellular proteins when the tissue pO2 is less than 10 mmHg. At 72 h after combined treatment of DU145-derived tumors, there was a significant decrease in the abundance of hypoxic fractions (Fig. 5a,b). In PC3-derived tumors, significant increases in the hypoxic fractions were observed at 24 h after irradiation treatment for both the XRT alone and the combined treatment groups compared with SQAP treatment alone (Fig. 5c,d).
Discussion The combination of α-SQMG and XRT was previously reported to inhibit the growth of solid tumors derived from human cancer cells that were implanted subcutaneously into nude mice.15,16 In the present study, we showed that SQAP could sensitize PCa cells to radiotherapy. We reported that the radiosensitization effect of SQAP in DU145 cells, which are androgen independent and relatively resistant to radiotherapy and apoptotic signals, could be partly mediated through suppression of angiogenesis and the collapse of normal tumor vessel structures, thereby enhancing normal oxygenation in the tumor and increasing radiosensitivity.18 Previous results have shown that anti-angiogenic effects contribute to the enhanced radioresponse in xenograft tumors after treatment with a combination of α-SQMG, a novel sulfoglycolipid, and two fractions of XRT.19 We also showed the effects of combined treatment with α-SQMG and 12-Gy doses of XRT on the remodeling of the tumor microenvironment using a human colon cancer cell line.16 Accumulating evidence supports that vascular normalization occurs during the early stages of the anti-angiogenic process and reduces hypoxic fractions, which prompted us to examine the possibility that radiosensitization occurs at the time of the second irradiation in the combined regimen.16,17 We found that radioresistant PCa (represented by DU145 cells) showed characteristic properties of vascular normalization, such as pericyte recruitment and reduced hypoxic fractions, at approximately 3 days after treatment with the combined regimen, when the second irradiation was administered. Preliminary data showed that the micronuclei yield was significantly higher (approximately twofold) in tumors that received 12 Gy at 72 h than in those that received the combined treatment at 6 h. This finding supported the importance of the second irradiation for radiosensitization in our
Fig. 4 Effects of SQAP and/or XRT on CD34 and -SMA in DU145 and PC3 xenografts. (a) DU145 and (c) PC3: representative images of microvessel (CD34: green) and pericyte (-SMA: red) staining in treated tumors at 6 and 72 h after initiating treatment. Microvessels covered by pericytes (yellow) are shown. (b) DU145 and (d) PC3: quantitative comparisons of CD34 and -SMA after initiating treatment. Each bar represents the mean ± SD of four different tumors. The mean of approximately 10 fields near the center of each tumor was assigned a representative value for each tumor. The relative density was normalized to the DAPI intensity. *P < 0.05. © 2015 The Japanese Urological Association
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Fig. 5 Effects of SQAP and/or XRT on the hypoxic fractions in DU145 and PC3 xenografts. (a) DU145 and (c) PC3: representative images of hypoxic fractions (green) at 6 or 72 h after treatment. (b) DU145 and (d) PC3: quantitative comparisons of hypoxic fractions after treatment. Each bar represents the mean ± SD of four different tumors. The mean of approximately four fields imaged near the center of each tumor was assigned a representative value for each tumor. The ratio was normalized to the DAPI intensity. *P < 0.05.
combined regimen, and suggested that remodeling of PCa in the DU145 microenvironment, including angiogenic and hypoxic characteristics, also contributed to the enhanced radiosensitivity. Regarding the appropriate timing for irradiation after SQAP treatment, another coworker investigated this using various times for irradiation (0.5, 1, 2 and 6 h after treatment with SQAP); these results showed that the most favorable timing was 0.5 h after SQAP treatment for inhibition of tumor growth (unpubl. data). The present findings provided evidence for the mechanism responsible for the enhanced radioresponse of this treatment regimen in PCa. The radiosensitization of oxygenated tumor tissues in DU145 cells to the irradiation as a result of vascular normalization has been shown to usually occur in the early stages; however, in the present study, it persisted until the late stages.17 Surprisingly, the effect of oxygenation in the tumor site was shown at 30 days after the combined treatment. These results suggested that the treatment using SQAP plus multiple fractions of XRT was appropriate and suitable for conventional radiotherapy for PCa, which is generally treated by irradiation with multiple fractions. However, the mechanisms mediating the long-term effects of oxygenation using XRT + SQAP are still unclear. Therefore, in future studies, we will examine the effects of SQAP plus multiple fractions of XRT on tumor growth in nude mice. Interestingly, PC3-derived tumors did not show the vascular normalization or reduction of hypoxic fractions, even though the tumor size was reduced, after combined treatment. Although the radiosensitivity of PC3 cells was higher than that of DU145 cells, the histological findings of PC3 cells at 3 days after the first XRT showed similar structures of tumor vessels compared with the observations in DU145 cells. Furthermore, we could not detect reduced hypoxic fractions in PC3-derived tumors treated with combine therapy. Therefore, the radiosensitizing effects of SQAP in vascular normalization could have different outcomes and depend on innate radiosensitivity in various tumors. 594
Current clinical reports have shown that combinations of anti-angiogenic agents and chemotherapeutic agents are unlikely to be highly effective, which might be attributed to insufficient optimization and timing of the combined therapy.19–21 Recent literature has shown that vascular normalization likely has a limited time window.17 Thus, it is important to identify biomarkers that can be used to monitor vascular normalization. As the mechanism of vascular normalization is still largely unknown, it is also possible that elucidating this mechanism will help determine the optimal window, which would potentially improve the timing for combined therapies. In addition, there are many molecules involved in the control and regulation of sensitivity to irradiation therapy in PCa, and elucidation of the molecular mechanisms underlying the enhanced radioresponse of SQAP is critical.22–26 SQAP has several characteristics. First, although the agent alone has no substantial effect on tumor growth at low concentrations, it induces radiosensitizing effects. By definition, agents with this property are called radiosensitizers.14 These types of agents could markedly reduce adverse effects, because they are typically injected systemically. Another benefit of SQAP is that it preferentially accumulates in tumors. If effective chemical compounds combining SQAP and boric acid can be synthesized, boric acid-mediated boron neutron capture therapy might be established in PCa.27,28 Thus, advancements in the field of radiation oncology that improve targeting of radiation to tumor tissues will further enhance the usefulness of this agent, particularly in PCa. In conclusion, SQAP has great potential for radiosensitization in PCa through its ability to remodel the tumor microenvironment, as shown by the induction of vascular normalization. However, further studies to elucidate the mechanisms involved in determining the radiosensitivity of PCa cells are required before this therapy can be implemented in clinical practice in humans. Furthermore, optimal therapeutic © 2015 The Japanese Urological Association
SQAP as a radiosensitizer in cancer
combinations and conditions required to achieve efficient outcomes must be determined.
Acknowledgment This study was supported by MEXT, the Support Program for the Strategic Research Foundation at Private Universities.
Conflict of interest None declared.
References 1 Baade PD, Youlden DR, Cramb SM, Dunn J, Gardiner RA. Epidemiology of prostate cancer in the Asia-Pacific region. Prostate Int. 2013; 1: 47–58. 2 Ward JF, Moul JW. Rising prostate-specific antigen after primary prostate cancer therapy. Nat. Clin. Pract. Urol. 2005; 2: 174–82. 3 Bolla M, Collette L, Blank L et al. Long-term results with immediate androgen suppression and external irradiation in patients with locally advanced prostate cancer (an EORTC study): a phase III randomised trial. Lancet 2002; 360: 103–6. 4 Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1995; 1: 27–31. 5 Kuenen BC, Levi M, Meijers JC et al. Potential role of platelets in endothelial damage observed during treatment with cisplatin, gemcitabine, and the angiogenesis inhibitor SU5416. J. Clin. Oncol. 2003; 21: 2192–8. 6 Mauceri HJ, Hanna NN, Beckett MA et al. Combined effects of angiostatin and ionizing radiation in antitumour therapy. Nature 1998; 394: 287–91. 7 Jastaniyah N, Murtha A, Pervez N et al. Phase I study of hypofractionated intensity modulated radiation therapy with concurrent and adjuvant temozolomide in patients with glioblastoma multiforme. Radiat. Oncol. 2013; 8: 38. 8 El Kaffas A, Al-Mahrouki A, Tran WT, Giles A, Czarnota GJ. Sunitinib effects on the radiation response of endothelial and breast tumor cells. Microvasc. Res. 2014; 92: 1–9. 9 Hottinger AF, Aissa AB, Espeli V et al. Phase I study of sorafenib combined with radiation therapy and temozolomide as first-line treatment of high-grade glioma. Br. J. Cancer 2014; 110: 2655–61. 10 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: 324–32. 11 Mizushina Y, Watanabe I, Ohta K et al. Studies on inhibitors of mammalian DNA polymerase α and β: sulfolipids from a pteridophyte, Athyrium niponicum. Biochem. Pharmacol. 1998; 55: 537–41. 12 Ohta K, Mizushina Y, Hirata N et al. Sulfoquinovosyldiacylglycerol, KM043, a new potent inhibitor of eukaryotic DNA polymerases and HIV-reverse transcriptase type 1 from a marine red alga, Gigartina tenella. Chem. Pharm. Bull. 1998; 46: 684–6.
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13 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: 85–92. 14 Murakami C, Yamazaki T, Hanashima S et al. A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)glyceride with stearic acid. Biochim. Biophys. Acta 2003; 1645: 72–80. 15 Sakimoto I, Ohta K, Yamazaki T et al. Alpha-sulfoquinovosylmonoacylglycerol is a novel potent radiosensitizer targeting tumor angiogenesis. Cancer Res. 2006; 66: 2287–95. 16 Ohta K, Murata H, Mori Y et al. Remodeling of the tumor microenvironment by combined treatment with a novel radiosensitizer, {alpha}sulfoquinovosylmonoacylglycerol ({alpha}-SQMG) and X-irradiation. Anticancer Res 2010; 30: 4397–404. 17 Jain RK. Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005; 307: 58–62. 18 Jayakumar S, Kunwar A, Sandur SK, Pandey BN, Chaubey RC. Differential response of DU145 and PC3 prostate cancer cells to ionizing radiation: role of reactive oxygen species, GSH and Nrf2 in radiosensitivity. Biochim. Biophys. Acta 2014; 1840: 485–94. 19 Vincenzi B, Santini D, Russo A et al. Bevacizumab in association with de Gramont 5-fluorouracil/folinic acid in patients with oxaliplatin-, irinotecan-, and cetuximab-refractory colorectal cancer: a single-center phase 2 trial. Cancer 2009; 115: 4849–56. 20 Hsu CH, Yang TS, Hsu C et al. Efficacy and tolerability of bevacizumab plus capecitabine as first-line therapy in patients with advanced hepatocellular carcinoma. Br. J. Cancer 2010; 102: 981–6. 21 Kindler HL, Niedzwiecki D, Hollis D et al. Gemcitabine plus bevacizumab compared with gemcitabine plus placebo in patients with advanced pancreatic cancer: phase III trial of the Cancer and Leukemia Group B (CALGB 80303). J. Clin. Oncol. 2010; 28: 3617–22. 22 Harada H, Itasaka S, Kizaka-Kondoh S et al. The Akt/mTOR pathway assures the synthesis of HIF-1alpha protein in a glucose- and reoxygenation-dependent manner in irradiated tumors. J. Biol. Chem. 2009; 284: 5332–42. 23 Chang L, Graham PH, Hao J et al. PI3K/Akt/mTOR pathway inhibitors enhance radiosensitivity in radioresistant prostate cancer cells through inducing apoptosis, reducing autophagy, suppressing NHEJ and HR repair pathways. Cell Death Dis. 2014; 5: e1437. 24 Bonkhoff H. Factors implicated in radiation therapy failure and radiosensitization of prostate cancer. Prostate Cancer 2012; 2012: 593241. 25 Ni X, Zhang Y, Ribas J et al. Prostate-targeted radiosensitization via aptamershRNA chimeras in human tumor xenografts. J. Clin. Invest. 2011; 121: 2383–90. 26 Han S, Brenner JC, Sabolch A et al. Targeted radiosensitization of ETS fusionpositive prostate cancer through PARP1 inhibition. Neoplasia 2013; 15: 1207–17. 27 Gifford I, Vreeland W, Grdanovska S et al. Liposome-based delivery of a boron-containing cholesteryl ester for high-LET particle-induced damage of prostate cancer cells: a boron neutron capture therapy study. Int. J. Radiat. Biol. 2014; 90: 480–5. 28 Yasui L, Kroc T, Gladden S, Andorf C, Bux S, Hosmane N. Boron neutron capture in prostate cancer cells. Appl. Radiat. Isot. 2012; 70: 6–12.
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doi: 10.1111/iju.12753
Original Article: Laboratory Investigation
Sulfoquinovosylacylpropanediol is a novel potent radiosensitizer in prostate cancer Yugo Sawada,1 Kazuya Omoto,1 Naoki Kohei,1 Kengo Sakaguchi,2 Masahiko Miura3 and Kazunari Tanabe1 1
Department of Urology, Tokyo Women’s Medical University, Tokyo, 2Department of Applied Biology, Faculty of Science and Technology, Tokyo University of Science, Chiba and 3Oral Radiation Oncology, Department of Oral Restitution, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan Abbreviations & Acronyms α-SQMG = α-sulfoquinovosylmonoacylglycerol DAPI = 4´6´-diamindino-2phenylindole dihydrochloride PBS = phosphate-buffered saline PCa = prostate cancer SMA = smooth muscle actin SQAP = sulfoquinovosylacylpropanediol XRT = X-ray irradiation Correspondence: Kazunari Tanabe M.D., Ph.D., Department of Urology, Tokyo Women’s Medical University, 8-1 Kawadacho, Shinjuku-ku, Tokyo 162-8666, Japan. Email: [email protected]. ac.jp Received 22 November 2014; accepted 11 February 2015. Online publication 17 March 2015
Objectives: To examine the effects of combined treatment with sulfoquinovosylacylpropanediol and X-ray irradiation on the remodeling of the prostate cancer microenvironment, including angiogenic and hypoxic characteristics. Methods: Human prostate cancer cells (DU145 and PC3) were implanted subcutaneously into the right hind legs of athymic nude mice. After the tumor volume reached 100–300 mm3, 2 mg/kg/day sulfoquinovosylacylpropanediol was given intravenously from day 0 to day 4, and cells were exposed to 4 Gy X-ray irradiation on days 0 and 3 (for a total of 8 Gy). Tumors were fixed and stained for pathological analyses and immunohistochemical evaluations. To analyze vascular normalization, 60 mg/kg pimonidazole dissolved in saline was injected intraperitoneally. Results: Combined treatment with sulfoquinovosylacylpropanediol plus X-ray irradiation enhanced growth inhibition in DU145 xenografts. The tumor vessel density in DU145 cells significantly decreased after the combined treatment. Staining for smooth muscle actin in vessels was significantly increased. Pimonidazole staining, showing hypoxic lesions, was negative from 72 h, but positive at 6 and 24 h after the first combined treatment. In contrast, no enhancement of the microenvironment in PC3 xenografts was observed with sulfoquinovosylacylpropanediol plus X-ray irradiation. Conclusion: Sulfoquinovosylacylpropanediol could be a novel potent radiosensitizing agent targeting angiogenesis in prostate cancer.
Key words:
oxygenation, prostate cancer, radiation therapy, radiosensitizer, vascular
normalization.
Introduction PCa is the second most common cause of cancer and the sixth leading cause of cancer death in men worldwide, with an estimated 1.1 million new cases and 307 000 deaths in 2012. The American Cancer Society estimated that 233 000 new cases of PCa will be diagnosed, and that 29 480 men will die of PCa in 2014. Besides skin cancer, PCa is the most common cancer and the second leading cause of cancer death in the USA. Interestingly, the incidence of PCa varies more than 25-fold among different countries worldwide. In Asian countries including Japan, the incidence of PCa continues to increase.1 Although the majority of patients with PCa can be successfully treated with surgery or radiation therapy, recurrence is observed in approximately 20–40% of patients within 10 years of treatment.2 The risk of recurrence is elevated to approximately 50% for patients with locally advanced PCa, a condition that is primarily treated by radiation therapy.3 At this stage in the disease, PCa cells are only moderately responsive or even unresponsive to the cytotoxic effects of chemotherapy or radiotherapy. Increased concentrations of high doses of radiation and cytotoxic agents fail to improve the response to therapy, conferring resistance to apoptosis in PCa cells. Therefore, it is essential to identify anticancer agents that are highly effective, non-toxic to normal cells and induce cell death preferentially in tumor cells. Inhibiting angiogenesis is a promising approach for the treatment of cancer, and various antiangiogenic agents have been developed.4 However, many anti-angiogenic agents are associated with adverse events, such as hemorrhage, thrombotic disease and hypertension.5 Importantly, monotherapy with anti-angiogenic agents is not expected to be an effective cure for cancer. Therefore, we have attempted to identify an effective cancer-specific treatment by combining
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SQAP as a radiosensitizer in cancer
anti-angiogenic agents and chemotherapeutic agents or irradiation. Previous studies have investigated the combined use of angiogenesis inhibitors and radiosensitizers, including hypoxic cell sensitizers; however, no therapeutic regimens have yet been used as standard treatments.6–9 SQAP is a sulfoglyco-lipid originally derived from sea urchins, and they are also naturally found in higher plants or sea algae (Fig. 1).10–12 Our group has established the complete chemical synthesis of SQAP.13,14 Sahara et al. reported that αSQMG, which is a prototype of SQAP, suppresses the growth of human solid tumors transplanted into nude mice, but shows no or little effect on in vitro cultured tumor cells.13 Furthermore, Sakimoto et al. reported that α-SQMG combined with radiation synergistically suppresses tumor growth at low doses that do not have inhibitory effects on tumor growth when administered alone.15 Ohta et al. reported that α-SQMG enhances the radioresponse of tumor tissue, accompanied by significant induction of vascular normalization, leading to oxygenation during radiation therapy.16 However, the effects of SQAP in PCa are not yet known. Therefore, in the present study, we examined the effects of combined SQAP and XRT in PCa cells.
(mm) × width (mm) × height (mm) × π / 6. This study was approved by the Animal Ethics Committee of the Faculty of Medicine, Tokyo Women’s Medical University.
Treatments Synthesized SQAP was prepared 1 day before experimentation and dissolved in saline at the appropriate concentrations. To analyze the tumor radioresponse, 2 mg/kg SQAP (dissolved in 100 μL of saline) was given intravenously to the SQAP group and the XRT + SQAP group five times daily from days 0–4. The same amount of saline was injected into mice in the control group and the XRT group. Non-anesthetized mice were irradiated (4 Gy/fraction) on days 0 and 3 with X-ray therapeutic machines (MBR-1520R-3; Hitati, Tokyo, Japan) while shielding the body with lead. Mice were killed, and the tumors were dissected and rapidly frozen on days 10 and 20. To analyze vascular normalization, the same treatments were carried out as for the tumor radioresponse, except that the mice were irradiated and given SQAP once on day 0, and 60 mg/kg pimonidazole dissolved in saline was injected intraperitoneally 30 min before the mice were killed at the indicated times after starting treatment.
Methods
Colony-forming assay
Cell lines and animal studies
Clonogenic cell survival was evaluated by dose–survival curves using colony-forming assays. Cells were irradiated in plastic tubes, plated in 60-mm dishes and incubated at 37 °C. After 7–10 days of incubation, cells were fixed and stained with crystal violet. Colonies containing more than 50 cells were counted. The surviving fraction is determined as follows. We counted the colonies plated in 60-mm dishes in which tumor cells were not irradiated before incubation. The plating efficiency was calculated as the number of colonies divided by the number of plated cells. Therefore, the ratio of the irradiated colonies to the total number of colonies was considered the survival fraction.
DU145 and PC3 human prostate cancer cells were, obtained from the American Type Culture Collection and maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin and 1 mmol/L sodium pyruvate at 37 °C in a 5% CO2 humidified atmosphere. Viable tumor cells (2 × 106) were implanted subcutaneously into the right hind legs of male BALB/c Slc-nude mice (6–8 weeks-of-age). After the tumor volume reached 100–300 mm3, the tumors were treated, and tumor growth was monitored by palpation. The size of the palpable tumors was measured using calipers every 2 days. The tumor volume (V, mm3) was estimated based on the length
Fig. 1 Chemical structure of SQAP. SQAP consists of three parts: glucose, propanediol and stearic acid. © 2015 The Japanese Urological Association
Immunofluorescence staining and image analysis To analyze the tumor radioresponse, consecutive 10-μm-thick tumor cryosections were fixed in 4% paraformaldehyde in PBS at room temperature, incubated in blocking buffer and then probed overnight at 4 °C with primary antibodies targeting endothelial cells (CD34; 1:100; Abcam Japan, Tokyo, Japan). After extensive washing in PBS, the sections were incubated with Alexa 488-conjugated secondary antibodies (1:200; Invitrogen, Carlsbad, CA, USA) for 30 min at room temperature. To examine vascular normalization, the cryosections were fixed in 4% formaldehyde, incubated in blocking buffer, and then probed with a Cy3-conjugated antibody specific for pericytes (α-SMA; 1:1000; Sigma, St. Louis, MO, USA) overnight at 4 °C. To detect hypoxia, the sections were fixed in icecold acetone for 10 min, and then incubated with an antipimonidazole primary antibody followed by an Alexa 488-conjugated secondary antibody according to the manufacturer’s instructions (Hypoxyprobe-1; Natural Pharmacia International, Burlington, MA, USA). After extensive washing in PBS, all of the immunostained sections were covered with an 591
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antifade agent containing DAPI (Vector Laboratories, Burlingame, CA, USA) and examined with a fluorescence microscope (Axio Observer Z1; Zeiss, Göttingen, Germany). Tumor sections were imaged using a 4× or 10× objective, digitized after background correction and then analyzed using Photoshop CS6 software (Adobe Systems, San Jose, CA, USA). Eight-bit grayscale images from adjacent microscopic fields were acquired and digitally overlaid for multiple stains. To quantify the stained areas, the grayscale images were converted to binary images, and the number of pixels was counted. The normalization index was calculated as the ratio of α-SMA to CD34. For all quantifications, the data were normalized based on the DAPI intensity, which was stained simultaneously with the specific markers.
Statistical analysis Mean values were statistically compared using Student’s t-test or ANOVA. Differences with P-values >0.05 were considered significant.
the cytotoxic activity of SQAP in Hela cells was only approximately 60% that of α-SQMG, which was used in previous experiments (data not shown).
Radiosensitivities of DU145 and PC3 cells differed Because we expected that the differences observed in Figure 2 might be a result of variations in the radiosensitivities of the cell lines, we examined the effects of XRT alone on cell survival using colony-forming assays. PC3 cells showed significantly increased radiosensitivity compared with DU145 cells in vitro (Fig. 3). Therefore, these data suggested that the radiosensitivity of PC3 cells was too strong to observe any synergistic effects of the combined therapy, as shown in Figure 2.
Results Combination of SQAP and radiation enhanced growth inhibition in DU145 xenografts In the present study, we used DU145 and PC3 prostate cancer cells, and established tumors in mice. Tumors derived from DU145 cells showed greater inhibition of tumor growth after combined treatment in vivo (Fig. 2a). However, tumors derived from PC3 cells showed the most growth inhibition in both the XRT group and the XRT + SQAP group, suggesting that the combined treatment had no synergistic effects in this cell line (Fig. 2b). Notably, SQAP alone did not significantly affect tumor growth in either cell line. Regarding the toxicity of SQAP, we confirmed that there were no side-effects in the mice receiving 20 mg/kg/day SQAP. In addition, MMT assays showed that
Fig. 3 Dose–survival curves in DU145 ( ) and PC3 ( ) cells after irradiation. The surviving fractions were determined by colony-forming assays. *P < 0.05.
Fig. 2 Relative tumor volumes over time. Five injections of SQAP (2 mg/kg, i.v.) plus two fractions of X-ray irradiation (4 Gy/fraction) were given. Vehicle ), SQAP ( ; 2 mg/kg/injection), XRT ( ; 4 Gy/ ( ) groups were analyzed. fraction) and XRT + SQAP ( Arrows indicate injections. Each point represents the mean ± SD of five or six tumors. *P < 0.05: XRT versus XRT plus SQAP.
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Synergistic effects of the combined treatment resulted in increased expression of α-SMA in tumor tissues We used dual staining for CD34 and α-SMA, markers of endothelial cells and pericytes, respectively, to determine whether tumor vascular normalization was induced with combined treatment. To this end, we examined the point at which pericytes were recruited around the tumor vessels after treatment as a characteristic marker of vascular normalization.17 The normalization index was determined as the ratio of the areas stained for α-SMA to those stained for CD34. Up to 24 h after treatment, regions stained for CD34 and α-SMA in DU145-derived tumors did not show remarkable changes. However, there was a significant increase in the normalization index in DU145-derived tumors, including an increase in α-SMA staining 72 h after combined treatment (Fig. 4a,b). In contrast, no changes in the vascular normalization index were observed for PC3-derived tumors (Fig. 4c,d).
Vascular normalization resulting from combined treatment caused decreased hypoxic regions in tumor tissues When vascular normalization is induced in tumor tissue, the hypoxic fraction is decreased.17 To analyze the hypoxic regions in DU145-derived tumors, we used pimonidazole, which forms adducts in cellular proteins when the tissue pO2 is less than 10 mmHg. At 72 h after combined treatment of DU145-derived tumors, there was a significant decrease in the abundance of hypoxic fractions (Fig. 5a,b). In PC3-derived tumors, significant increases in the hypoxic fractions were observed at 24 h after irradiation treatment for both the XRT alone and the combined treatment groups compared with SQAP treatment alone (Fig. 5c,d).
Discussion The combination of α-SQMG and XRT was previously reported to inhibit the growth of solid tumors derived from human cancer cells that were implanted subcutaneously into nude mice.15,16 In the present study, we showed that SQAP could sensitize PCa cells to radiotherapy. We reported that the radiosensitization effect of SQAP in DU145 cells, which are androgen independent and relatively resistant to radiotherapy and apoptotic signals, could be partly mediated through suppression of angiogenesis and the collapse of normal tumor vessel structures, thereby enhancing normal oxygenation in the tumor and increasing radiosensitivity.18 Previous results have shown that anti-angiogenic effects contribute to the enhanced radioresponse in xenograft tumors after treatment with a combination of α-SQMG, a novel sulfoglycolipid, and two fractions of XRT.19 We also showed the effects of combined treatment with α-SQMG and 12-Gy doses of XRT on the remodeling of the tumor microenvironment using a human colon cancer cell line.16 Accumulating evidence supports that vascular normalization occurs during the early stages of the anti-angiogenic process and reduces hypoxic fractions, which prompted us to examine the possibility that radiosensitization occurs at the time of the second irradiation in the combined regimen.16,17 We found that radioresistant PCa (represented by DU145 cells) showed characteristic properties of vascular normalization, such as pericyte recruitment and reduced hypoxic fractions, at approximately 3 days after treatment with the combined regimen, when the second irradiation was administered. Preliminary data showed that the micronuclei yield was significantly higher (approximately twofold) in tumors that received 12 Gy at 72 h than in those that received the combined treatment at 6 h. This finding supported the importance of the second irradiation for radiosensitization in our
Fig. 4 Effects of SQAP and/or XRT on CD34 and -SMA in DU145 and PC3 xenografts. (a) DU145 and (c) PC3: representative images of microvessel (CD34: green) and pericyte (-SMA: red) staining in treated tumors at 6 and 72 h after initiating treatment. Microvessels covered by pericytes (yellow) are shown. (b) DU145 and (d) PC3: quantitative comparisons of CD34 and -SMA after initiating treatment. Each bar represents the mean ± SD of four different tumors. The mean of approximately 10 fields near the center of each tumor was assigned a representative value for each tumor. The relative density was normalized to the DAPI intensity. *P < 0.05. © 2015 The Japanese Urological Association
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Fig. 5 Effects of SQAP and/or XRT on the hypoxic fractions in DU145 and PC3 xenografts. (a) DU145 and (c) PC3: representative images of hypoxic fractions (green) at 6 or 72 h after treatment. (b) DU145 and (d) PC3: quantitative comparisons of hypoxic fractions after treatment. Each bar represents the mean ± SD of four different tumors. The mean of approximately four fields imaged near the center of each tumor was assigned a representative value for each tumor. The ratio was normalized to the DAPI intensity. *P < 0.05.
combined regimen, and suggested that remodeling of PCa in the DU145 microenvironment, including angiogenic and hypoxic characteristics, also contributed to the enhanced radiosensitivity. Regarding the appropriate timing for irradiation after SQAP treatment, another coworker investigated this using various times for irradiation (0.5, 1, 2 and 6 h after treatment with SQAP); these results showed that the most favorable timing was 0.5 h after SQAP treatment for inhibition of tumor growth (unpubl. data). The present findings provided evidence for the mechanism responsible for the enhanced radioresponse of this treatment regimen in PCa. The radiosensitization of oxygenated tumor tissues in DU145 cells to the irradiation as a result of vascular normalization has been shown to usually occur in the early stages; however, in the present study, it persisted until the late stages.17 Surprisingly, the effect of oxygenation in the tumor site was shown at 30 days after the combined treatment. These results suggested that the treatment using SQAP plus multiple fractions of XRT was appropriate and suitable for conventional radiotherapy for PCa, which is generally treated by irradiation with multiple fractions. However, the mechanisms mediating the long-term effects of oxygenation using XRT + SQAP are still unclear. Therefore, in future studies, we will examine the effects of SQAP plus multiple fractions of XRT on tumor growth in nude mice. Interestingly, PC3-derived tumors did not show the vascular normalization or reduction of hypoxic fractions, even though the tumor size was reduced, after combined treatment. Although the radiosensitivity of PC3 cells was higher than that of DU145 cells, the histological findings of PC3 cells at 3 days after the first XRT showed similar structures of tumor vessels compared with the observations in DU145 cells. Furthermore, we could not detect reduced hypoxic fractions in PC3-derived tumors treated with combine therapy. Therefore, the radiosensitizing effects of SQAP in vascular normalization could have different outcomes and depend on innate radiosensitivity in various tumors. 594
Current clinical reports have shown that combinations of anti-angiogenic agents and chemotherapeutic agents are unlikely to be highly effective, which might be attributed to insufficient optimization and timing of the combined therapy.19–21 Recent literature has shown that vascular normalization likely has a limited time window.17 Thus, it is important to identify biomarkers that can be used to monitor vascular normalization. As the mechanism of vascular normalization is still largely unknown, it is also possible that elucidating this mechanism will help determine the optimal window, which would potentially improve the timing for combined therapies. In addition, there are many molecules involved in the control and regulation of sensitivity to irradiation therapy in PCa, and elucidation of the molecular mechanisms underlying the enhanced radioresponse of SQAP is critical.22–26 SQAP has several characteristics. First, although the agent alone has no substantial effect on tumor growth at low concentrations, it induces radiosensitizing effects. By definition, agents with this property are called radiosensitizers.14 These types of agents could markedly reduce adverse effects, because they are typically injected systemically. Another benefit of SQAP is that it preferentially accumulates in tumors. If effective chemical compounds combining SQAP and boric acid can be synthesized, boric acid-mediated boron neutron capture therapy might be established in PCa.27,28 Thus, advancements in the field of radiation oncology that improve targeting of radiation to tumor tissues will further enhance the usefulness of this agent, particularly in PCa. In conclusion, SQAP has great potential for radiosensitization in PCa through its ability to remodel the tumor microenvironment, as shown by the induction of vascular normalization. However, further studies to elucidate the mechanisms involved in determining the radiosensitivity of PCa cells are required before this therapy can be implemented in clinical practice in humans. Furthermore, optimal therapeutic © 2015 The Japanese Urological Association
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combinations and conditions required to achieve efficient outcomes must be determined.
Acknowledgment This study was supported by MEXT, the Support Program for the Strategic Research Foundation at Private Universities.
Conflict of interest None declared.
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