Cancer Res Treat.  2018 Oct;50(4):1362-1377. 10.4143/crt.2017.353.

Preclinical Study of Novel Curcumin Analogue SSC-5 Using Orthotopic Tumor Xenograft Model for Esophageal Squamous Cell Carcinoma

• Tung LN
• Song S
• Chan KT
• Choi MY
• Lam HY
• Chan CM
• Chen Z
• Wang HK
• Leung HT
• Law S
• Huang Y
• Song H
• Lee NP

• KMID: 2424808
• DOI: http://doi.org/10.4143/crt.2017.353

Abstract

PURPOSE
Tumor xenograft model is an indispensable animal cancer model. In esophageal squamous cell carcinoma (ESCC) research, orthotopic tumor xenograft model establishes tumor xenograft in the animal esophagus, which allows the study of tumorigenesis in its native microenvironment.
MATERIALS AND METHODS
In this study,we described two simple and reproducible methods to develop tumor xenograft at the cervical or the abdominal esophagus in nude mice by direct injection of ESCC cells in the esophageal wall.
RESULTS
In comparing these two methods, the cervical one presented with more clinically relevant features, i.e., esophageal stricture, body weight loss and poor survival. In addition, the derived tumor xenografts accompanied a rapid growth rate and a high tendency to invade into the surrounding structures. This model was subsequently used to study the anti-tumor effect of curcumin, which is known for its potential therapeutic effects in various diseases including cancers, and its analogue SSC-5. SSC-5 was selected among the eight newly synthesized curcumin analogues based on its superior anti-tumor effect demonstrated in an MTT cell proliferation assay and its effects on apoptosis induction and cell cycle arrest in cultured ESCC cells. Treatment of orthotopic tumor-bearing mice with SSC-5 resulted in an inhibition in tumor growth and invasion.
CONCLUSION
Taken together, we have established a clinically relevant orthotopic tumor xenograft model that can serve as a preclinical tool for screening new anti-tumor compounds, e.g., SSC-5, in ESCC.

Keyword

Curcumin; Esophageal squamous cell carcinoma

MeSH Terms

Animals
Apoptosis
Body Weight
Carcinogenesis
Carcinoma, Squamous Cell*
Cell Cycle Checkpoints
Cell Proliferation
Curcumin*
Epithelial Cells*
Esophageal Stenosis
Esophagus
Heterografts*
Mass Screening
Mice
Mice, Nude
Therapeutic Uses
Curcumin
Therapeutic Uses

Figure

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  Fig. 1. Surgical procedures to inject esophageal squamous cell carcinoma (ESCC) cells at different anatomical locations in the mouse esophagus. ESCC cells mixed with Matrigel were injected into the esophageal wall at the cervical esophagus (A) and at the abdominal esophagus (B).

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  Fig. 2. Comparison between orthotopic tumor xenograft models at the cervical and abdominal esophagus. (A) Photos taken at week 4 (cervical) or week 6 (abdominal) after SLMT-1 esophageal squamous cell carcinoma cell injection into the esophageal wall show successful establishment of tumor xenografts, marked by asterisk, at the cervical or abdominal esophagus. (B) Mice bearing tumor xenograft at the cervical esophagus experienced a significant body weight reduction in 4 weeks’ time, whereas a gradual increase was noticed in those with tumor xenograft at the abdominal esophagus. Bars indicate mean±standard deviation (SD). **p < 0.01. (C) When the tumor xenografts were dissected for tumor size measurement every two weeks, we observed faster growth of tumor xenografts at the cervical esophagus compared to those at the abdominal esophagus. Error bars indicate SD. *p < 0.05. ns, statistically not significant. (D) Kaplan-Meier survival curve analysis revealed poorer survival in mice bearing tumor xenograft at the cervical esophagus than those with tumor xenograft at the abdominal esophagus. p < 0.001. n, animal number.

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  Fig. 3. Histological images of SLMT-1-derived tumor xenografts at the cervical and abdominal esophagus. (A) Mouse esophagus with tumor xenograft was dissected to prepare serial sections from proximal location S1 to distal location S4, as indicated by dotted lines. (B) Hematoxylin and eosin staining was used to reveal the histology of the representative sections at location S1, S2, S3, and S4. S1, no observable tumor; S2, tumor centered in the muscularis propria and submucosa of the esophageal wall; S3 and S4, tumor invasion into the esophageal lumen and at the same time causing luminal stricture (×40). (C) The tumor invasive front can be clearly seen in a high magnification image of squared area in S3 section (×200). E, epithelium of esophagus; L, esophageal lumen; M, muscular layer of esophagus; T, tumor.

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  Fig. 4. Immunohistochemical staining of Ki-67 and CD34 in orthotopic tumor xenografts. Tumor xenografts formed at the cervical and abdominal esophagus using SLMT-1 esophageal squamous cell carcinoma cells were subjected to immunohistochemistry by staining with two different molecular markers, i.e., Ki-67 for proliferating cells (A) and CD34 for tumor vessels (B). Most of the positively stained cells (arrows) were concentrated at the tumor periphery (area F) rather than the tumor center (area C).

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  Fig. 5. Molecular structures of curcumin and its analogues. Curcumin, 1,7-Bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; SSC-3, 3,5-Bis-(4-chlorobenzylidene)-1-methylpiperidin-4-one; SSC-5, 1-Methyl-3,5-bis-(3,4,5-trimethoxybenzylidene)piperidin-4-one; SSC-6, 3,5-Bis-(4-hydroxy-3-methoxybenzylidene)-1-methylpiperidin-4-one; SSC-8, 1-Methyl-3, 5-bispyridin-4-ylmethylenepiperidin-4-one; SSC-9, 1-Methyl-3,5-bispyridin-3-ylmethylenepiperidin-4-one; SSC-10, 3,5-Bis-(4-fluorobenzylidene)-1-methylpiperidin-4-one; SSC-19, 3,5-Bis-(3-hydroxybenzylidene)-1-methylpiperidin-4-one; SSC-20, 3,5-Bis-(2,5-dihydroxybenzylidene)-1-methylpiperidin-4-one.

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  Fig. 6. Curcumin analogues exhibit potent growth inhibiting effect in cultured esophageal squamous cell carcinoma (ESCC) cells. In vitro MTT cell proliferation assay was performed in three ESCC cell lines SLMT-1, HKESC-2, and KYSE-270 after treatment with curcumin (0-20 μM) and its analogues (0-5 μM). Based on the IC50 obtained after a 48-hour treatment period, curcumin analogues demonstrated more potent growth inhibiting effect in ESCC cells than curcumin. Representative results are shown.

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  Fig. 7. SSC-5 and SSC-19 induce cell cycle arrest and apoptosis in cultured esophageal squamous cell carcinoma (ESCC) cells. (A) Flow cytometry analysis revealed the DNA content of SLMT-1 and KYSE-450 ESCC cells after treatment with dissolved in dimethyl sulfoxide (control), curcumin, SSC-5, and SSC-19. Representative flow cytometric profiles are shown. Treatment with SSC-5 and SSC-19 resulted in a significant induction of cells in the sub-G1 and G2/M phase of the cell cycle. Bars indicate mean±standard deviation. *p < 0.05, **p < 0.01. (B) Western blotting experiment showed an obvious induction of cleaved poly(ADPribose) polymerase (PARP) and a de-phosphorylation of Bcl-2 in ESCC cells after treatment with SSC-5 and SSC-19. β-Actin was used as a loading control.

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  Fig. 8. SSC-5 inhibits growth and invasion abilities of orthotopic tumor xenografts. Tumor xenografts were established at the cervical esophagus in nude mice using SLMT-1 and KYSE-450 esophageal squamous cell carcinoma (ESCC) cells. Tumorbearing mice were randomly divided into three groups for a 2-week treatment with dissolved in dimethyl sulfoxide (control), curcumin and SSC-5. (A) Treatment with SSC-5 resulted in a significant tumor size reduction (left panel) and a drop in the percentage of mice with tumor invasion into the esophageal lumen (middle panel). Right panel shows the representative photographs from each experimental group. Data are presented in mean±standard error of mean. ns, statistically not significant. *p < 0.05. (B) Representative histological images show tumor invasion into the esophageal lumen in control group, while the occurrence of such event was reduced under the treatment with curcumin or SSC-5 (×40). E, epithelium of esophagus; L, esophageal lumen; M, muscular layer of esophagus; T, tumor.

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  Fig. 9. SSC-5 treatment induces expression level of cleaved poly (ADP-ribose) polymerase (PARP) in orthotopic tumor xenografts. KYSE-450 esophageal squamous cell carcinoma cells were used to establish tumor xenografts at the cervical esophagus in nude mice. Treatment of these mice with SSC-5 induced the expression level of cleaved PARP in tumor xenografts, while a lower level was detected in tumor xenografts from curcumin treatment and control group.

Reference

References

1. Killion JJ, Radinsky R, Fidler IJ. Orthotopic models are necessary to predict therapy of transplantable tumors in mice. Cancer Metastasis Rev. 1998; 17:279–84.

2. Fidler IJ, Wilmanns C, Staroselsky A, Radinsky R, Dong Z, Fan D. Modulation of tumor cell response to chemotherapy by the organ environment. Cancer Metastasis Rev. 1994; 13:209–22.

3. Dong Z, Radinsky R, Fan D, Tsan R, Bucana CD, Wilmanns C, et al. Organ-specific modulation of steady-state mdr gene expression and drug resistance in murine colon cancer cells. J Natl Cancer Inst. 1994; 86:913–20.

4. Hori T, Yamashita Y, Ohira M, Matsumura Y, Muguruma K, Hirakawa K. A novel orthotopic implantation model of human esophageal carcinoma in nude rats: CD44H mediates cancer cell invasion in vitro and in vivo. Int J Cancer. 2001; 92:489–96.

5. Ohara T, Takaoka M, Sakurama K, Nagaishi K, Takeda H, Shirakawa Y, et al. The establishment of a new mouse model with orthotopic esophageal cancer showing the esophageal stricture. Cancer Lett. 2010; 293:207–12.

6. Kuroda S, Kubota T, Aoyama K, Kikuchi S, Tazawa H, Nishizaki M, et al. Establishment of a non-invasive semi-quantitative bioluminescent imaging method for monitoring of an orthotopic esophageal cancer mouse model. PLoS One. 2014; 9:e114562.

7. Song S, Chang D, Cui Y, Hu J, Gong M, Ma K, et al. New orthotopic implantation model of human esophageal squamous cell carcinoma in athymic nude mice. Thorac Cancer. 2014; 5:417–24.

8. Hu T, Qi H, Li P, Zhao G, Ma Y, Hao Q, et al. Comparison of GFP-expressing imageable mouse models of human esophageal squamous cell carcinoma established in various anatomical sites. Anticancer Res. 2015; 35:4655–63.

9. Ip JC, Ko JM, Yu VZ, Chan KW, Lam AK, Law S, et al. A versatile orthotopic nude mouse model for study of esophageal squamous cell carcinoma. Biomed Res Int. 2015; 2015:910715.

10. Padhye S, Chavan D, Pandey S, Deshpande J, Swamy KV, Sarkar FH. Perspectives on chemopreventive and therapeutic potential of curcumin analogs in medicinal chemistry. Mini Rev Med Chem. 2010; 10:372–87.

11. Bandyopadhyay D. Farmer to pharmacist: curcumin as an anti-invasive and antimetastatic agent for the treatment of cancer. Front Chem. 2014; 2:113.

12. Wei X, Du ZY, Cui XX, Verano M, Mo RQ, Tang ZK, et al. Effects of cyclohexanone analogues of curcumin on growth, apoptosis and NF-kappaB activity in PC-3 human prostate cancer cells. Oncol Lett. 2012; 4:279–84.

13. Kunnumakkara AB, Guha S, Krishnan S, Diagaradjane P, Gelovani J, Aggarwal BB. Curcumin potentiates antitumor activity of gemcitabine in an orthotopic model of pancreatic cancer through suppression of proliferation, angiogenesis, and inhibition of nuclear factor-kappaB-regulated gene products. Cancer Res. 2007; 67:3853–61.

14. Seo JA, Kim B, Dhanasekaran DN, Tsang BK, Song YS. Curcumin induces apoptosis by inhibiting sarco/endoplasmic reticulum Ca2+ ATPase activity in ovarian cancer cells. Cancer Lett. 2016; 371:30–7.

15. Liu Z, Xie Z, Jones W, Pavlovicz RE, Liu S, Yu J, et al. Curcumin is a potent DNA hypomethylation agent. Bioorg Med Chem Lett. 2009; 19:706–9.

16. Nagaraju GP, Zhu S, Wen J, Farris AB, Adsay VN, Diaz R, et al. Novel synthetic curcumin analogues EF31 and UBS109 are potent DNA hypomethylating agents in pancreatic cancer. Cancer Lett. 2013; 341:195–203.

17. Meiyanto E, Putri DD, Susidarti RA, Murwanti R, Sardjiman , Fitriasari A, et al. Curcumin and its analogues (PGV-0 and PGV-1) enhance sensitivity of resistant MCF-7 cells to doxorubicin through inhibition of HER2 and NF-kB activation. Asian Pac J Cancer Prev. 2014; 15:179–84.

18. Lai KK, Chan KT, Choi MY, Wang HK, Fung EY, Lam HY, et al. 14-3-3sigma confers cisplatin resistance in esophageal squamous cell carcinoma cells via regulating DNA repair molecules. Tumour Biol. 2016; 37:2127–36.

19. Lee NP, Chan KT, Choi MY, Lam HY, Tung LN, Tzang FC, et al. Oxygen carrier YQ23 can enhance the chemotherapeutic drug responses of chemoresistant esophageal tumor xenografts. Cancer Chemother Pharmacol. 2015; 76:1199–207.

20. Costi R, Di Santo R, Artico M, Miele G, Valentini P, Novellino E, et al. Cinnamoyl compounds as simple molecules that inhibit p300 histone acetyltransferase. J Med Chem. 2007; 50:1973–7.

21. Wei X, Du ZY, Zheng X, Cui XX, Conney AH, Zhang K. Synthesis and evaluation of curcumin-related compounds for anticancer activity. Eur J Med Chem. 2012; 53:235–45.

22. Yadav B, Taurin S, Rosengren RJ, Schumacher M, Diederich M, Somers-Edgar TJ, et al. Synthesis and cytotoxic potential of heterocyclic cyclohexanone analogues of curcumin. Bioorg Med Chem. 2010; 18:6701–7.

23. Bazzaro M, Anchoori RK, Mudiam MK, Issaenko O, Kumar S, Karanam B, et al. α,β-Unsaturated carbonyl system of chalcone-based derivatives is responsible for broad inhibition of proteasomal activity and preferential killing of human papilloma virus (HPV) positive cervical cancer cells. J Med Chem. 2011; 54:449–56.

24. Qiu X, Liu Z, Shao WY, Liu X, Jing DP, Yu YJ, et al. Synthesis and evaluation of curcumin analogues as potential thioredoxin reductase inhibitors. Bioorg Med Chem. 2008; 16:8035–41.

25. Chan KT, Choi MY, Lai KK, Tan W, Tung LN, Lam HY, et al. Overexpression of transferrin receptor CD71 and its tumorigenic properties in esophageal squamous cell carcinoma. Oncol Rep. 2014; 31:1296–304.

26. Zhang L, Zhang X, Barrisford GW, Olumi AF. Lexatumumab (TRAIL-receptor 2 mAb) induces expression of DR5 and promotes apoptosis in primary and metastatic renal cell carcinoma in a mouse orthotopic model. Cancer Lett. 2007; 251:146–57.

27. Abou-Elkacem L, Arns S, Brix G, Gremse F, Zopf D, Kiessling F, et al. Regorafenib inhibits growth, angiogenesis, and metastasis in a highly aggressive, orthotopic colon cancer model. Mol Cancer Ther. 2013; 12:1322–31.

28. Wilmanns C, Fan D, Obrian C, Radinsky R, Bucana C, Tsan R, et al. Modulation of doxorubicin sensitivity and level of p-glycoprotein expression in human colon-carcinoma cells by ectopic and orthotopic environments in nude-mice. Int J Oncol. 1993; 3:413–22.

29. Kuo TH, Kubota T, Watanabe M, Furukawa T, Kase S, Tanino H, et al. Site-specific chemosensitivity of human small-cell lung carcinoma growing orthotopically compared to subcutaneously in SCID mice: the importance of orthotopic models to obtain relevant drug evaluation data. Anticancer Res. 1993; 13:627–30.

30. Jiang YJ, Lee CL, Wang Q, Zhou ZW, Yang F, Jin C, et al. Establishment of an orthotopic pancreatic cancer mouse model: cells suspended and injected in Matrigel. World J Gastroenterol. 2014; 20:9476–85.

31. Quatromoni JG, Predina JD, Bhojnagarwala P, Judy RP, Jiang J, De Jesus EM, et al. Adenoviral-based immunotherapy provides local disease control in an orthotopic murine model of esophageal cancer. J Immunother. 2014; 37:283–92.

32. Tian F, Zhang C, Tian W, Jiang Y, Zhang X. Comparison of the effect of p65 siRNA and curcumin in promoting apoptosis in esophageal squamous cell carcinoma cells and in nude mice. Oncol Rep. 2012; 28:232–40.

33. Qiu X, Du Y, Lou B, Zuo Y, Shao W, Huo Y, et al. Synthesis and identification of new 4-arylidene curcumin analogues as potential anticancer agents targeting nuclear factor-kappaB signaling pathway. J Med Chem. 2010; 53:8260–73.

34. Zhou J, Geng G, Shi Q, Sauriol F, Wu JH. Design and synthesis of androgen receptor antagonists with bulky side chains for overcoming antiandrogen resistance. J Med Chem. 2009; 52:5546–50.