Teng-Long-Bu-Zhong-Tang, a Chinese herbal formula, enhances anticancer effects of 5--Fluorouracil in CT26 colon carcinoma

Shan Deng, Bing Hu, Hong-Mei An, Qin Du, Ling Xu, Ke-Ping Shen, Xiu-Feng Shi, Meng-Meng Wei, Yang Wu, Shan Deng, Bing Hu, Hong-Mei An, Qin Du, Ling Xu, Ke-Ping Shen, Xiu-Feng Shi, Meng-Meng Wei, Yang Wu

Abstract

Background: Colorectal cancer remains one of the leading causes of cancer death worldwide. Traditional Chinese Medicine (TCM) has played a positive role in colorectal cancer treatment. There is a great need to establish effective herbal formula for colorectal cancer treatment. Based on TCM principles and clinical practices, we have established an eight herbs composed formula for colorectal cancer treatment, which is Teng-Long-Bu-Zhong-Tang (TLBZT). We have demonstrated the anticancer effects of TLBZT against colorectal carcinoma in vitro. In present study, we evaluated the anticancer potential of TLBZT, used alone or in combination with low dose of 5-Fluorouracil (5-Fu), in CT26 colon carcinoma in vivo.

Methods: CT26 colon carcinoma was established in BALB/c mice and treated with TLBZT, 5-Fu, or TLBZT plus 5-Fu. The tumor volumes were observed. Apoptosis was detected by TUNEL assay. Caspases activities were detected by colorimetric assay. Cell senescence was indentified by senescence β-galactosidase staining. Gene expression and angiogenesis was observed by immunohistochemistry or western blot.

Results: TLBZT significantly inhibited CT26 colon carcinoma growth. TLBZT elicited apoptosis in CT26 colon carcinoma, accompanied by Caspase-3, 8, and 9 activation and PARP cleavage, and downregulation of XIAP and Survivin. TLBZT also induced cell senescence in CT26 colon carcinoma, with concomitant upregulation of p16 and p21 and downregulation of RB phosphorylation. In addition, angiogenesis and VEGF expression in CT26 colon carcinoma was significantly inhibited by TLBZT treatment. Furthermore, TLBZT significantly enhanced anticancer effects of 5-Fu in CT26 colon carcinoma.

Conclusions: TLBZT exhibited significantly anticancer effect, and enhanced the effects of 5-Fu in CT26 colon carcinoma, which may correlate with induction of apoptosis and cell senescence, and angiogenesis inhibition. The present study provides new insight into TCM approaches for colon cancer treatment that are worth of further study.

Figures

Figure 1
Figure 1
TLBZT and 5-FU inhibited CT26 carcinoma growth. Female BALB/c mice were injected s.c. with 1 × 106 CT26 cells. When the tumors were palpable, the mice were randomized to receive treatment with TLBZT, 5-Fu, TLBZT plus 5-Fu, or distilled water as a control. Tumor volumes were monitored every three days (A). After three weeks of treatment, the tumors were removed and weighed (B). **P<0.01, versus control group, ##P<0.01, versus TLBZT or 5-Fu group.
Figure 2
Figure 2
TLBZT and 5-FU induced apoptosis in CT26 carcinoma. After three weeks treatment, CT26 carcinomas were collected and subjected to TUNEL assay (3 tumors/group) and observed under microscope (×200) (A). The apoptotic cells were counted in 3 high power fields (HPF) in each slide. The percentage of apoptosis cells were expressed as means ± SD (B). **P<0.01, versus control group. ##P<0.01, versus TLBZT group, ΔP<0.05, versus 5-Fu group.
Figure 3
Figure 3
TLBZT and 5-FU activitied Caspases. After three weeks treatment, CT26 carcinomas (3 tumors/group) were removed, homogenized, and subjected to Caspase-3, Caspase-8 and Caspase-9 activities assay by cleavage of specific substrate. Caspases activities were expressed as fold activation over control (A). Cleavage of PARP was detected by immunohistochemistry (3 tumors/group) and observed under microscope (×200) (B). Cleaved PARP positive cells (3 HPF/slide) were counted by Image-Pro Plus 6.0 software and expressed as percentage of total cells (C). D, PARP Cleavage in CT26 carcinomas were further verified with western blot by antibody against cleavage PARP (c-PARP). GAPDH was used as a loading control. **P<0.01, versus control group, ##P<0.01, versus TLBZT or 5-Fu group.
Figure 4
Figure 4
Effects of TLBZT and 5-FU on XIAP and Survivin expression. After three weeks treatment, expression of XIAP and Survivin in CT26 carcinoma (3 tumors/group) were detected by immunohistochemistry and observed under microscope (×200) (A). The mean optical density (MOD) of XIAP and Survivin (3 HPF/slide) were analyzed by Image-Pro Plus 6.0 software, and expressed as fold of control (B). C, expression of XIAP and Survivin in CT26 carcinomas were further verified with western blot by specific antibody. GAPDH was used as a loading control. **P<0.01, versus control group, ##P<0.01, versus TLBZT or 5-Fu group.
Figure 5
Figure 5
Effects of TLBZT and 5-FU on cell senescence in CT26 carcinoma. A, after three weeks treatment, CT26 carcinomas (3 tumors/group) were removed and frozen cut into 7μm sections, fixed, reacted with SA-β-gal staining solution overnight and observed under microscope (×200). B, SA-β-gal positive staining was counted in 3 HPF in each slide and expressed as fold over control. **P<0.01, versus control group, ##P<0.01, versus TLBZT group, #P<0.05, versus TLBZT group.
Figure 6
Figure 6
Effects of TLBZT on cell senescence related gene expression. After three weeks treatment, p16 and p21, and RB phosphorylation in CT26 carcinomas (3 tumors/group) were detected by immunohistochemistry and observed under microscope (×200) (A). The p21, p16 and RB phosphorylation positive cells (3 HPF/slide) were counted by Image-Pro Plus 6.0 software, and expressed as mean ± SD (B). C, p16 and p21 expression and RB phosphorylation was further detected with western blot by specific antibody. GAPDH was used as a loading control. **P<0.01, versus control group.
Figure 7
Figure 7
Effects of TLBZT and 5-FU on angiogenesis. A, after three weeks treatment, angiogenesis and VEGF expression in CT26 carcinomas (3 tumors/group) were detected by immunohistochemistry and observed under microscope (×200). B, vessel density was determined via counting the number of the microvessels per high-power field (3 HPF/slide), and the MOD of VEGF (3 HPF/slide) were analyzed by Image-Pro Plus 6.0 software, and expressed as fold over control. C, VEGF expression in CT26 carcinomas were further verified by western blot. GAPDH was used as a loading control. **P<0.01, versus control group. ▲P>0.05, versus control group.

References

    1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90. doi: 10.3322/caac.20107.
    1. Storli KE, Søndenaa K, Bukholm IR, Nesvik I, Bru T, Furnes B, Hjelmeland B, Iversen KB, Eide GE. Overall survival after resection for colon cancer in a national cohort study was adversely affected by TNM stage, lymph node ratio, gender, and old age. Int J Colorectal Dis. 2011;26(10):1299–1307. doi: 10.1007/s00384-011-1244-2.
    1. Davies JM, Goldberg RM. Treatment of metastatic colorectal cancer. Semin Oncol. 2011;38(4):552–560. doi: 10.1053/j.seminoncol.2011.05.009.
    1. Deng S, Hu B, An HM. Traditional Chinese Medicinal Syndromes and Treatment in Colorectal Cancer. J Cancer Ther. 2012;3(6):888–897. doi: 10.4236/jct.2012.326114.
    1. Hu B, Du Q, Shen KP, Xu L. Principles and Scientific Basis of Traditional Chinese Medicine in Cancer Treatment. J Bioanal Biomed. 2012;S6:005. doi: 10.4172/1948-593X.S6-005.
    1. Deng S, Hu B, Shen KP. Pathogenesis and treatment of colorectal cancer in Traditional Chinese Medicine. World Sci Technol Mod Trad Chinese Med Mater Med. 2012;14(4):1858–1862. Chinese.
    1. Wang XR, Yuan XB, Li P, Gao L. The TCM Syndrome Distribution in Colorectal Cancer. J Changchun Coll Trad Chinese Med. 2011;2(3):377–379. Chinese.
    1. Hu B, An HM, Shen KP, Du Q. Effects of Tenglong Buzhong Decoction on proliferation and apoptosis of human colon carcinoma cell line LS-174T. J Chinese Integr Med. 2010;8(6):575–580. doi: 10.3736/jcim20100611. Chinese.
    1. Hu B, An HM, Shen KP, Du Q. Senescence-inducing effects of Chinese herbal medicine Tenglong Buzhong Decoction on human colon carcinoma LS-174T cells and the mechanism. J Chinese Integr Med. 2010;8(11):1048–1052. doi: 10.3736/jcim20101108. Chinese.
    1. Wong RS. Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer Res. 2011;26(30):87.
    1. Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG, Earnshaw WC. Cleavage of poly (ADP-ribose) polymerase by a proteinase with properties like ICE. Nature. 1994;371(6495):346–347. doi: 10.1038/371346a0.
    1. Xiang G, Wen X, Wang H, Chen K, Liu H. Expression of X-linked inhibitor of apoptosis protein in human colorectal cancer and its correlation with prognosis. J Surg Oncol. 2009;100(8):708–712. doi: 10.1002/jso.21408.
    1. Hernandez JM, Farma JM, Coppola D, Hakam A, Fulp WJ, Chen DT, Siegel EM, Yeatman TJ, Shibata D. Expression of the antiapoptotic protein survivin in colon cancer. Clin Colorectal Cancer. 2011;10(3):188–193. doi: 10.1016/j.clcc.2011.03.014.
    1. Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O, Peacocke M, Campisi J. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA. 1995;92(20):9363–9367. doi: 10.1073/pnas.92.20.9363.
    1. Roninson IB. Tumor cell senescence in cancer treatment. Cancer Res. 2003;63(11):2705–2715.
    1. Zhang NN, Bu P, Zhu HH, Shen WG. Inhibitory effects of Scutellaria barbatae D. Don on tumor angiogenesis and its mechanism. Chinese J cancer. 2005;24(12):1459–1463. Chinese.
    1. Wei L, Lin J, Xu W, Cai Q, Shen A, Hong Z, Peng J. Scutellaria barbata D. Don Inhibits Tumor Angiogenesis via Suppression of Hedgehog Pathway in a Mouse Model of Colorectal Cancer. Int J Mol Sci. 2012;13(8):9419–9430.
    1. Elluru SR, Duong Van Huyen JP, Delignat S, Prost F, Heudes D, Kazatchkine MD, Friboulet A, Kaveri SV. Antiangiogenic properties of viscum album extracts are associated with endothelial cytotoxicity. Anticancer Res. 2009;29(8):2945–2950.
    1. Van Huyen JP, Bayry J, Delignat S, Gaston AT, Michel O, Bruneval P, Kazatchkine MD, Nicoletti A, Kaveri SV. Induction of apoptosis of endothelial cells by Viscum album: a role for anti-tumoral properties of mistletoe lectins. Mol Med. 2002;8(10):600–6.
    1. Kuzu I, Bicknell R, Harris AL, Jones M, Gatter KC, Mason DY. Heterogeneity of vascular endothelial cells with relevance to diagnosis of vascular tumours. J Clin Pathol. 1992;45(2):143–148. doi: 10.1136/jcp.45.2.143.
    1. Chetty C, Lakka SS, Bhoopathi P, Kunigal S, Geiss R, Rao JS. Tissue inhibitor of metalloproteinase 3 suppresses tumor angiogenesis in matrix metalloproteinase 2-down-regulated lung cancer. Cancer Res. 2008;68(12):4736–4745. doi: 10.1158/0008-5472.CAN-07-6612.
    1. Hsieh HY, Chiu PH, Wang SC. Epigenetics in traditional chinese pharmacy: a bioinformatic study at pharmacopoeia scale. Evid Based Complement Alternat Med. 2011;201(1):816714.
    1. Zuo LL, Wang ZY, Fan ZL, Tian SQ, Liu JR. Evaluation of Antioxidant and Antiproliferative Properties of Three Actinidia (Actinidia kolomikta, Actinidia arguta, Actinidia chinensis) Extracts in Vitro. Int J Mol Sci. 2012;13(5):5506–5518.
    1. Chen YJ, Shi RJ. Ethanol extract from radix of Actinidia chinensis inhibits cell proliferation and induces apoptosis in human colon carcinoma cell line LoVo. World Chinese J Digestol. 2012;20(18):1657–1661. Chinese.
    1. Ding X, Zhu FS, Li M, Gao SG. Induction of apoptosis in human hepatoma SMMC-7721 cells by solamargine from Solanum nigrum L. J Ethnopharmacol. 2012;139(2):599–604. doi: 10.1016/j.jep.2011.11.058.
    1. Ji YB, Gao SY. Study on Mitochondrion Pathway of the Apoptosis of HepG2 Induced by Solanine. Chinese Pharm J. 2008;43(3):272–275.
    1. Ji YB, Yuan HC, Gao SY, Sun HB. Material basis of cytotoxicity in polysaccharide from Solanum nigrum. Chinese Trad Herbal Drugs. 2011;42(11):2275–2278. Chinese.
    1. Nawab A, Thakur VS, Yunus M, Ali Mahdi A, Gupta S. Selective cell cycle arrest and induction of apoptosis in human prostate cancer cells by a polyphenol-rich extract of Solanum nigrum. Int J Mol Med. 2012;29(2):277–284.
    1. Peng B, Hu Q, Liu X, Wang L, Chang Q, Li J, Tang J, Wang N, Wang Y. Duchesnea phenolic fraction inhibits in vitro and in vivo growth of cervical cancer through induction of apoptosis and cell cycle arrest. Exp Biol Med (Maywood) 2009;234(1):74–83. doi: 10.3181/0806-RM-204.
    1. Peng B, Chang Q, Wang L, Hu Q, Wang Y, Tang J, Liu X. Suppression of human ovarian SKOV-3 cancer cell growth by Duchesnea phenolic fraction is associated with cell cycle arrest and apoptosis. Gynecol Onco. 2008;108(1):173–181. doi: 10.1016/j.ygyno.2007.09.016.
    1. Zhu QJ, Zheng GJ, Zhang D. Study on antitumor effect and mechanism of extract of Atractylodes macrocephala Koidz. J Shandong Univ Trad Chinese Med. 2006;30(1):69–71. Chinese.
    1. Kikuchi T, Uchiyama E, Ukiya M, Tabata K, Kimura Y, Suzuki T, Akihisa T. Cytotoxic and apoptosis-inducing activities of triterpene acids from Poria cocos. J Nat Prod. 2011;74(2):137–144. doi: 10.1021/np100402b.
    1. Gapter L, Wang Z, Glinski J, Ng KY. Induction of apoptosis in prostate cancer cells by pachymic acid from Poria cocos. Biochem Biophys Res Commun. 2005;332(4):1153–1161. doi: 10.1016/j.bbrc.2005.05.044.
    1. Lu Y, Li CS, Dong Q. Chinese herb related molecules of cancer-cell-apoptosis: a minireview of progress between Kanglaite injection and related genes. J Exp Clin Cancer Res. 2008;27:31. doi: 10.1186/1756-9966-27-31.
    1. Chung CP, Hsu CY, Lin JH, Kuo YH, Chiang W, Lin YL. Antiproliferative lactams and spiroenone from adlay bran in human breast cancer cell lines. J Agric Food Chem. 2011;59(4):1185–94. doi: 10.1021/jf104088x.
    1. Goh D, Lee YH, Ong ES. Inhibitory effects of a chemically standardized extract from Scutellaria barbata in human colon cancer cell lines. LoVo J Agric Food Chem. 2005;53(21):8197–8204. doi: 10.1021/jf051506+.
    1. Wang SM, Ye M, Ni ZM. Effect of mistletoe lectins on proliferation and apoptosis in HT-29 colon cancer cell. Chinese J Clin Pharmacol Ther. 2007;12(9):1028–1031. Chinese.
    1. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26:239–257. doi: 10.1038/bjc.1972.33.
    1. Brown JM, Attardi LD. The role of apoptosis in cancer development and treatment response. Nat Rev Cancer. 2005;5:231.
    1. Miura K, Fujibuchi W, Ishida K, Naitoh T, Ogawa H, Ando T, Yazaki N, Watanabe K, Haneda S, Shibata C, Sasaki I. Inhibitor of apoptosis protein family as diagnostic markers and therapeutic targets of colorectal cancer. Surg Today. 2011;41(2):175–182. doi: 10.1007/s00595-010-4390-1.
    1. Connolly K, Mitter R, Muir M, Jodrell D, Guichard S. Stable XIAP knockdown clones of HCT116 colon cancer cells are more sensitive to TRAIL, taxanes and irradiation in vitro. Cancer Chemother Pharmacol. 2009;64(2):307–316. doi: 10.1007/s00280-008-0872-x.
    1. Cai M, Wang GB, Tao KX, Cai CX. Enhanced chemotherapy sensitivity of human colon cancer cells to 5-fluorouracil by siRNA recombinant expression vector targeting survivin gene. Chin Med Sci J. 2009;24(2):97–101. doi: 10.1016/S1001-9294(09)60069-9.
    1. Chiantore MV, Vannucchi S, Mangino G, Percario ZA, Affabris E, Fiorucci G, Romeo G. Senescence and cell death pathways and their role in cancer therapeutic outcome. Curr Med Chem. 2009;16(3):287–300. doi: 10.2174/092986709787002691.
    1. Gewirtz DA, Holt SE, Elmore LW. Accelerated senescence: an emerging role in tumor cell response to chemotherapy and radiation. Biochem Pharmacol. 2008;76(8):947–957. doi: 10.1016/j.bcp.2008.06.024.
    1. Nardella C, Clohessy JG, Alimonti A, Pandolfi PP. Pro-senescence therapy for cancer treatment. Nat Rev Cancer. 2011;11(7):503–511. doi: 10.1038/nrc3057.
    1. Chang UM, Li CH, Lin LI, Huang CP, Kan LS, Lin SB. Ganoderiol F, a ganoderma triterpene, induces senescence in hepatoma HepG2 cells. Life Sci. 2006;79(12):1129–1139. doi: 10.1016/j.lfs.2006.03.027.
    1. Peng CC, Chen KC, Peng RY, Chyau CC, Su CH, Hsieh-Li HM. Antrodia camphorata extract induces replicative senescence in superficial TCC, and inhibits the absolute migration capability in invasive bladder carcinoma cells. J Ethnopharmacol. 2007;109(1):93–103. doi: 10.1016/j.jep.2006.07.009.
    1. Hu B, An HM, Shen KP, Xu L, DU Q, Deng S, Wu Y. Liver Yin deficiency tonifying herbal extract induces apoptosis and cell senescence in Bel-7402 human hepatocarcinoma cells. Exp Ther Med. 2012;3(1):80–86.
    1. Folkman J. Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971;285(21):1182–1186. doi: 10.1056/NEJM197111182852108.
    1. Rapisarda A, Melillo G. Role of the VEGF/VEGFR axis in cancer biology and therapy. Adv Cancer Res. 2012;114:237–267.
    1. Allavena P, Sica A, Garlanda C, Mantovani A. The Yin-Yang of tumor-associated macrophages in neoplastic progression and immune surveillance. Immunol Rev. 2008;222:155–161. doi: 10.1111/j.1600-065X.2008.00607.x.
    1. Zhu WJ, Yu DH, Zhao M, Lin MG, Lu Q, Wang QW, Guan YY, Li GX, Luan X, Yang YF, Qin XM, Fang C, Yang GH, Chen HZ. Antiangiogenic triterpenes isolated from Chinese herbal medicine Actinidia Chinensis Planch. Anticancer Agents Med Chem. 2012;13(2):195–198.
    1. Xu Y, Pan RL, Chang Q, Qin M, Liu Y, Tang JT. Experimental study of Solanum nigrum on inhibiting angiogenesis in chick chorioallantoic membrane. China J Chinese Mater Med. 2008;33(5):549–552. Chinese.
    1. Xu Y, Pan RL, Chang Q, Tang JT, Liu Y. Inhibitory effects of Duchesnea indica (andr) Focke on angiogenesis in chick chorioallantoic membrane. Pharmacol Clin Chinese Mater Med. 2008;24(3):65–67. Chinese.

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