Aspirin minimized the pro-metastasis effect of sorafenib and improved survival by up-regulating HTATIP2 in hepatocellular carcinoma

Lu Lu, Hui-Chuan Sun, Wei Zhang, Zong-Tao Chai, Xiao-Dong Zhu, Ling-Qun Kong, Wen-Quan Wang, Ke-Zhi Zhang, Yuan-Yuan Zhang, Qiang-Bo Zhang, Jian-Yang Ao, Jia-Qi Li, Lu Wang, Wei-Zhong Wu, Zhao-You Tang, Lu Lu, Hui-Chuan Sun, Wei Zhang, Zong-Tao Chai, Xiao-Dong Zhu, Ling-Qun Kong, Wen-Quan Wang, Ke-Zhi Zhang, Yuan-Yuan Zhang, Qiang-Bo Zhang, Jian-Yang Ao, Jia-Qi Li, Lu Wang, Wei-Zhong Wu, Zhao-You Tang

Abstract

Background aims: We previously demonstrated the pro-metastasis effect of sorafenib in hepatocellular carcinoma (HCC), which is mediated by down-regulation of tumor suppressor HTATIP2. The aim of the present study was to determine whether aspirin minimizes this effect and improves survival.

Methods: The effects of sorafenib, aspirin, and combined sorafenib and aspirin were observed in HCCLM3 and HepG2 xenograft nude mice. Tumor growth, intrahepatic metastasis (IHM), lung metastasis, and survival were assessed. Polymerase chain reaction (PCR) array, real-time (RT)-PCR, and Western blotting were used to examine gene expression. The anti-invasion and anti-metastasis effects of aspirin were studied in HTATIP2-knockdown and HTATIP2-overexpressing HCC cell lines. The molecular mechanism of HTATIP2 regulation by aspirin was explored.

Results: Aspirin suppressed the pro-invasion and pro-metastasis effects of sorafenib in HCC and up-regulated HTATIP2 expression. Aspirin did not inhibit the proliferation of HCC cells, but it decreased the invasiveness of HCC with lower expression of HTATIP2 and increased expression of a set of markers, indicating a mesenchymal-to-epithelial transition in tumor cells. The up-regulation of HTATPI2 expression by aspirin is most likely mediated through inhibition of cyclooxygenase (COX) 2 expression.

Conclusions: Aspirin minimized the pro-metastasis effect of sorafenib by up-regulating the tumor suppressor HTATIP2; this mechanism is mediated through inhibition of COX2.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Aspirin suppressed the pro-invasion and…
Figure 1. Aspirin suppressed the pro-invasion and pro-metastasis effects of sorafenib in orthotopic HCC models.
(A) Median survival was improved by sorafenib treatment with or without aspirin in orthotopic HCCLM3 and HepG2 models. Moreover, sorafenib combined with aspirin significantly prolonged median survival compared to sorafenib alone (98.5±3.85 days vs 81.0±4.31 days in the HCCLM3-wt model, P = 0.0076; 92.3±3.82 days vs 77.7±3.54 days in the HepG2-wt model, P = 0.0122). (B) In the HCCLM3-wt model (left panel), tumor size was 1.70±0.12 cm3 in the sorafenib group (P<0.001), 1.27±0.08 cm3 in the group treated with both sorafenib and aspirin (P<0.001), and 3.68±0.18 cm3 in the control group. In the HepG2-wt model (right panel), tumors were smaller in the sorafenib group (1.02±0.11 cm3, P<0.001), and the group treated with combined sorafenib and aspirin (0.84±0.11 cm3, P<0.001), compared with controls (2.90±0.29 cm3). (C) In the HCCLM3-wt model (left panel), the sorafenib group had more intrahepatic metastases (IHM), although tumor size was smaller, and the group treated with combined sorafenib and aspirin had significantly fewer IHM. The HepG2-wt model (right panel), was characterized by invasive growth, and sorafenib did not further increase its invasiveness and metastasis. However, tumor margins became clearer in the group treated with combined sorafenib and aspirin. (D) In the HCCLM3-wt model, there were obviously fewer lung metastases in the sorafenib group (9.67±1.71) compared to the control group (17.67±1.94, P = 0.0129) The incidence of lung metastasis (6/6) was not decreased in the sorafenib group. However, the standardized number of lung metastases (SNLM) was greater in the sorafenib group (5.72±0.24) than in the control group (4.77±0.33, P = 0.0457). In the group treated with combined aspirin and sorafenib, the incidence of lung metastasis (4/6) and SNLM were distinctly decreased compared with the group treated with sorafenib alone (2.84±1.04 vs 5.72±0.24, P = 0.0421). (E) Hematoxylin-eosin staining confirmed that sorafenib induced more IHM in the HCCLM3-wt model, and aspirin was able to reverse the adverse effect. In addition, aspirin was able to effect a clearer margin in the HepG2-wt model. (F) Comparison of IHM in the HCCLM3-wt model. The number of IHM was greater in the sorafenib group compared to the control group (6.00±1.65 vs 0.50±0.34, P = 0.0225) and lower in the sorafenib and aspirin combined therapy group compared to the sorafenib group (0.17±0.17 vs 6.00±1.65, P = 0.0171).
Figure 2. Aspirin up-regulated HTATIP2, which was…
Figure 2. Aspirin up-regulated HTATIP2, which was down-regulated by sorafenib.
(A) Gene network analysis by Ariadne software shows differences in 84 genes between the sorafenib and aspirin combination group and the sorafenib group. (B) Comparison of gene expression in the sorafenib and aspirin combination group and the sorafenib group. (C, D) Expression of HTATIP2 in HCCLM3-wt and HepG2-wt tumors was evaluated at the mRNA level (C) and protein level (D). (E, F) Expression of HTATIP2 in HCCLM3-wt and HepG2-wt cell lines was confirmed at the mRNA level (E) and protein level (F). (Columns, mean of 6 samples in each group; bars, SEM).
Figure 3. Up-regulation of HTATIP2 by aspirin…
Figure 3. Up-regulation of HTATIP2 by aspirin reversed epithelial-to-mesenchymal transition (EMT) and inhibited invasion in HCC cell lines.
(A) Expression of HTATIP2 mRNA was detected by RT-PCR (columns, mean of 6 samples in each group; bars, SEM; *, P<0.05). (B) Invasion of HCC cell lines with different expression levels of HTATIP2 was measured by transwell assay. Left panel: migrated tumor cells. Right panel: quantification of invasion assay. (C) Levels of HTATIP2 protein and EMT markers, including E-cadherin, N-cadherin, and vimentin, were revealed by Western blotting.
Figure 4. Aspirin up-regulated HTATIP2 expression and…
Figure 4. Aspirin up-regulated HTATIP2 expression and suppressed invasiveness and metastatic potential in a xenograft hepatoma model.
(A, B) Expression of HTATIP2 protein was detected by immunohistochemistry (A, original magnification, ×200) and Western blotting (B). (C) In HCCLM3-wt and HCCLM3-LV-shHTAITP2 models (left panel), HTATIP2 knockdown apparently increased IHM; aspirin did not affect these 2 models. In HepG2-wt and HepG2-LV-HTATIP2 models (right panel), overexpression of HTATIP2 obviously inhibited tumor invasion. The tumor margin was clearer with aspirin treatment in the HepG2-wt model. (D) Hematoxylin-eosin staining confirmed that lower expression of HTATIP2 promoted invasiveness and metastasis potential of HCC, which could be reserved by aspirin.
Figure 5. Aspirin up-regulated HTATIP2 by inhibiting…
Figure 5. Aspirin up-regulated HTATIP2 by inhibiting COX2 expression.
(A) Western blotting revealed that aspirin down-regulated COX1 and COX2 expression in HCC cell lines. (B) After 12 hours of treatment, Western blotting revealed a down-regulation of COX2 expression by aspirin, and NS-398 corresponded with an increase in HTATIP2. (C) The up-regulation of HTATIP2 mRNA by aspirin and NS-398 was confirmed by real-time PCR (columns, mean of 6 samples in each group; bars, SEM; *, P<0.05).

References

    1. Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin 55: 74–108.
    1. Poon D, Anderson BO, Chen LT, Tanaka K, Lau WY, et al. (2009) Management of hepatocellular carcinoma in Asia: consensus statement from the Asian Oncology Summit 2009. Lancet Oncol 10: 1111–1118.
    1. Tang ZY (2005) Hepatocellular carcinoma surgery–review of the past and prospects for the 21st century. J Surg Oncol 91: 95–96.
    1. Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, et al. (2008) Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 359: 378–390.
    1. Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, et al. (2009) Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol 10: 25–34.
    1. Desar IM, Mulder SF, Stillebroer AB, van Spronsen DJ, van der Graaf WT, et al. (2009) The reverse side of the victory: flare up of symptoms after discontinuation of sunitinib or sorafenib in renal cell cancer patients. A report of three cases. Acta Oncol 48: 927–931.
    1. Cacheux W, Boisserie T, Staudacher L, Vignaux O, Dousset B, et al. (2008) Reversible tumor growth acceleration following bevacizumab interruption in metastatic colorectal cancer patients scheduled for surgery. Ann Oncol 19: 1659–1661.
    1. Allegra CJ, Yothers G, O’Connell MJ, Sharif S, Petrelli NJ, et al. (2011) Phase III trial assessing bevacizumab in stages II and III carcinoma of the colon: results of NSABP protocol C-08. J Clin Oncol 29: 11–16.
    1. Powles T, Blank C, Chowdhury S, Horenblas S, Peters J, et al. (2011) The outcome of patients treated with sunitinib prior to planned nephrectomy in metastatic clear cell renal cancer. Eur Urol 60: 448–454.
    1. Ebos JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG, et al. (2009) Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell 15: 232–239.
    1. Paez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, et al. (2009) Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 15: 220–231.
    1. Zhang W, Sun HC, Wang WQ, Zhang QB, Zhuang PY, et al... (2012) Sorafenib Down-regulates Expression of HTATIP2 to Promote Invasiveness and Metastasis of Orthotopic Hepatocellular Carcinoma Tumors in Mice. Gastroenterology.
    1. Rothwell PM, Wilson M, Elwin CE, Norrving B, Algra A, et al. (2010) Long-term effect of aspirin on colorectal cancer incidence and mortality: 20-year follow-up of five randomised trials. Lancet 376: 1741–1750.
    1. Rothwell PM, Fowkes FG, Belch JF, Ogawa H, Warlow CP, et al. (2011) Effect of daily aspirin on long-term risk of death due to cancer: analysis of individual patient data from randomised trials. Lancet 377: 31–41.
    1. Rothwell PM, Wilson M, Price JF, Belch JF, Meade TW, et al. (2012) Effect of daily aspirin on risk of cancer metastasis: a study of incident cancers during randomised controlled trials. Lancet 379: 1591–1601.
    1. Saad-Hossne R, Prado RG, Hossne WS (2007) Effects of acetylsalicylic acid and acetic acid solutions on VX2 liver carcinoma in rabbits. In vivo analysis. Acta Cir Bras 22: 299–308.
    1. Abiru S, Nakao K, Ichikawa T, Migita K, Shigeno M, et al. (2002) Aspirin and NS-398 inhibit hepatocyte growth factor-induced invasiveness of human hepatoma cells. Hepatology 35: 1117–1124.
    1. Rothwell PM, Price JF, Fowkes FG, Zanchetti A, Roncaglioni MC, et al. (2012) Short-term effects of daily aspirin on cancer incidence, mortality, and non-vascular death: analysis of the time course of risks and benefits in 51 randomised controlled trials. Lancet 379: 1602–1612.
    1. Holmes MD, Chen WY, Li L, Hertzmark E, Spiegelman D, et al. (2010) Aspirin intake and survival after breast cancer. J Clin Oncol 28: 1467–1472.
    1. Li Y, Tian B, Yang J, Zhao L, Wu X, et al. (2004) Stepwise metastatic human hepatocellular carcinoma cell model system with multiple metastatic potentials established through consecutive in vivo selection and studies on metastatic characteristics. J Cancer Res Clin Oncol 130: 460–468.
    1. Zhao J, Lu B, Xu H, Tong X, Wu G, et al. (2008) Thirty-kilodalton Tat-interacting protein suppresses tumor metastasis by inhibition of osteopontin transcription in human hepatocellular carcinoma. Hepatology 48: 265–275.
    1. Zhao J, Chen J, Lu B, Dong L, Wang H, et al. (2008) TIP30 induces apoptosis under oxidative stress through stabilization of p53 messenger RNA in human hepatocellular carcinoma. Cancer Res 68: 4133–4141.
    1. Zhang W, Zhu XD, Sun HC, Xiong YQ, Zhuang PY, et al. (2010) Depletion of tumor-associated macrophages enhances the effect of sorafenib in metastatic liver cancer models by antimetastatic and antiangiogenic effects. Clin Cancer Res 16: 3420–3430.
    1. Chang YS, Adnane J, Trail PA, Levy J, Henderson A, et al. (2007) Sorafenib (BAY 43–9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models. Cancer Chemother Pharmacol 59: 561–574.
    1. Langley RE, Burdett S, Tierney JF, Cafferty F, Parmar MK, et al. (2011) Aspirin and cancer: has aspirin been overlooked as an adjuvant therapy? Br J Cancer 105: 1107–1113.
    1. de Groot JF, Fuller G, Kumar AJ, Piao Y, Eterovic K, et al. (2010) Tumor invasion after treatment of glioblastoma with bevacizumab: radiographic and pathologic correlation in humans and mice. Neuro Oncol 12: 233–242.
    1. Yu C, Friday BB, Lai JP, Yang L, Sarkaria J, et al. (2006) Cytotoxic synergy between the multikinase inhibitor sorafenib and the proteasome inhibitor bortezomib in vitro: induction of apoptosis through Akt and c-Jun NH2-terminal kinase pathways. Mol Cancer Ther 5: 2378–2387.
    1. Carter CA, Chen C, Brink C, Vincent P, Maxuitenko YY, et al. (2007) Sorafenib is efficacious and tolerated in combination with cytotoxic or cytostatic agents in preclinical models of human non-small cell lung carcinoma. Cancer Chemother Pharmacol 59: 183–195.
    1. De Bock K, Mazzone M, Carmeliet P (2011) Antiangiogenic therapy, hypoxia, and metastasis: risky liaisons, or not? Nat Rev Clin Oncol 8: 393–404.
    1. Casanovas O, Hicklin DJ, Bergers G, Hanahan D (2005) Drug resistance by evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet tumors. Cancer Cell 8: 299–309.
    1. Keunen O, Johansson M, Oudin A, Sanzey M, Rahim SA, et al. (2011) Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma. Proc Natl Acad Sci U S A 108: 3749–3754.
    1. Grivennikov SI, Greten FR, Karin M (2010) Immunity, inflammation, and cancer. Cell 140: 883–899.
    1. Dinarello CA (2010) Anti-inflammatory Agents: Present and Future. Cell 140: 935–950.
    1. Sitia G, Aiolfi R, Di Lucia P, Mainetti M, Fiocchi A, et al. (2012) Antiplatelet therapy prevents hepatocellular carcinoma and improves survival in a mouse model of chronic hepatitis B. Proc Natl Acad Sci U S A. 109: E2165–2172.
    1. Sahasrabuddhe VV, Gunja MZ, Graubard BI, Trabert B, Schwartz LM, et al... (2012) Nonsteroidal Anti-inflammatory Drug Use, Chronic Liver Disease, and Hepatocellular Carcinoma. J Natl Cancer Inst.
    1. Wu CY, Chen YJ, Ho HJ, Hsu YC, Kuo KN, et al. (2012) Association between nucleoside analogues and risk of hepatitis B virus-related hepatocellular carcinoma recurrence following liver resection. JAMA 308: 1906–1914.
    1. Hial V, De Mello MC, Horakova Z, Beaven MA (1977) Antiproliferative activity of anti-inflammatory drugs in two mammalian cell culture lines. J Pharmacol Exp Ther 202: 446–454.
    1. Futakuchi M, Ogawa K, Sano M, Tamano S, Takeshita F, et al. (2002) Suppression of lung metastasis by aspirin but not indomethacin in an in vivo model of chemically induced hepatocellular carcinoma. Jpn J Cancer Res 93: 1175–1181.
    1. Futakuchi M, Ogawa K, Tamano S, Takahashi S, Shirai T (2004) Suppression of metastasis by nuclear factor kappaB inhibitors in an in vivo lung metastasis model of chemically induced hepatocellular carcinoma. Cancer Sci 95: 18–24.
    1. Zhao J, Zhang X, Shi M, Xu H, Jin J, et al. (2006) TIP30 inhibits growth of HCC cell lines and inhibits HCC xenografts in mice in combination with 5-FU. Hepatology 44: 205–215.
    1. Hossain MA, Kim DH, Jang JY, Kang YJ, Yoon JH, et al. (2012) Aspirin enhances doxorubicin-induced apoptosis and reduces tumor growth in human hepatocellular carcinoma cells in vitro and in vivo. Int J Oncol 40: 1636–1642.
    1. Raza H, John A, Benedict S (2011) Acetylsalicylic acid-induced oxidative stress, cell cycle arrest, apoptosis and mitochondrial dysfunction in human hepatoma HepG2 cells. Eur J Pharmacol 668: 15–24.
    1. Baigent C, Blackwell L, Collins R, Emberson J, Godwin J, et al. (2009) Aspirin in the primary and secondary prevention of vascular disease: collaborative meta-analysis of individual participant data from randomised trials. Lancet 373: 1849–1860.
    1. Morgan G (2009) Aspirin for the primary prevention of vascular events? Public Health 123: 787–788.
    1. Huang ES, Strate LL, Ho WW, Lee SS, Chan AT (2011) Long-term use of aspirin and the risk of gastrointestinal bleeding. Am J Med 124: 426–433.
    1. Reagan-Shaw S, Nihal M, Ahmad N (2008) Dose translation from animal to human studies revisited. FASEB J 22: 659–661.
    1. Chan FK, Ching JY, Hung LC, Wong VW, Leung VK, et al. (2005) Clopidogrel versus aspirin and esomeprazole to prevent recurrent ulcer bleeding. N Engl J Med 352: 238–244.
    1. Chan FK, Chung SC, Suen BY, Lee YT, Leung WK, et al. (2001) Preventing recurrent upper gastrointestinal bleeding in patients with Helicobacter pylori infection who are taking low-dose aspirin or naproxen. N Engl J Med 344: 967–973.
    1. Lai KC, Lam SK, Chu KM, Wong BC, Hui WM, et al. (2002) Lansoprazole for the prevention of recurrences of ulcer complications from long-term low-dose aspirin use. N Engl J Med 346: 2033–2038.
    1. Yusuf S, Zhao F, Mehta SR, Chrolavicius S, Tognoni G, et al. (2001) Effects of clopidogrel in addition to aspirin in patients with acute coronary syndromes without ST-segment elevation. N Engl J Med 345: 494–502.

Source: PubMed

Sponsor e collaboratori

Condizioni mediche

Interventi farmacologici

3
Sottoscrivi