A NT I-BRE A ST CA NCE R A C T I V I T Y O F SP G - 5 6 F R O M S W E E T P O TA T O I N MC F- 7 BEARINGMI CE IN SIT U T HR O U G H P R O M O TI N G A P O P T O S I S A N D I N H I B I T I N G M ETAS TAS I SAUTHORSZhaoxing Li1,2, Yang Yu1, Meimei Wang1, Heshan Xu3, Bing Han1, Pu Jiang1,Hang Ma1, Yuanfeng Li3, Cheng Tian1, Deqi Zhou4, Xuegang Li1 & Xiaoli Ye3(2019) 1School of Pharmaceutical Sciences, Southwest University,Chongqing, 400716, China. 2McLean Hospital,nHarvard Medical School,Belmont, 02478, MA, USA. 3Chongqing Engineering Research Centre forSweet Potato, School of Life Sciences, Southwest University, Chongqing,400715, China. 4Oncology Department, Chongqing Beibei District Hospital of
Traditional Chinese Medicine, Chongqing, 400700, China. Zhaoxing Li andYang Yu contributed equally.

INTRODUCTION
Breast cancer is the second leading cause of cancer-related deaths aroundthe world, as well as the most common type of cancer in China. Induction ofapoptosis is one of the most important mechanisms by which an anti-tumor
agent works. Apoptosis can be induced via two pathways, an extrinsic deathsignal-induced receptor-mediated pathway, or an intrinsic distress-induced
mitochondrion-mediated pathway. Modern research results show that thesweet potato (Ipomoea batatas (L.) Lam.) and its active ingredients have theeffect of enhancing immunity, anti-oxidation, inhibiting inflammation andinhibiting cardiovascular diseases. Anti-cancer ingredients in sweet potatoes

mainly include polysaccharides, sporamin, DHEA, anthocyanin andglycoprotein. As a type of mucoprotein, sweet potato glycoprotein is an
important biological active constituent of sweet potatoes, with healthpromoting functions and medicinal properties. Glycoprotein havepharmacologic actions such as anti-cancer, anti-oxidant, anti-hyperlipidemia,
hypoglycaemic and immunological enhancement. Recently, Wang et al.
extracted and isolated sweet potato glycoprotein-56 (SPG-56) at molecularweight of 56000 which consists of 2.9% sugar and 97.1% protein, and wasfound to enhance the apoptosis of colon cancer cells.

OBJECTIVEIn this study, nude mice were used as experimental animals to evaluate theanti-breast cancer effects of SPG-56 in vivo and in vitro. To clarify its possible
molecular mechanisms, the effects on serum inflammation, tumor markers,
and related proteins were investigated. The results are expected to provide atheoretical and experimental basis for the use of SPG-56 in anti-breast
cancer applications in the future.

MATERIAL & METHODS
50 healthy four-week-old female BALB/c nude mice were randomly divided
into five groups, comprising normal controls (NC), tumor controls (TC), SPG56 with a low dose of 60 mg/kg body weight (SPG-L), SPG-56 with a middle
dose of 120 mg/kg body weight (SPG-M), and SPG 56 with a high dose of 240
mg/kg body weight (SPG-H). Prepared MCF-7 cell suspensions (30 μlMatrigel and 90 μl of PBS containing 106 cells) were orthotopically implanted
into the nude mouse breast pad. From the first day of inoculation, mice in theNC and TC groups received appropriate amounts of 0.9% saline solution.
SPG-56 at doses of 60, 120 and 240 mg/kg of body weight was administereddaily by gavage.
Luminescence Imaging for Detection of Metastasis in Vivo.
Using the same animal breeds and feeding methods described above, 12
4T1-Luc breast cancer bearing mice were randomly divided into two groups,
4T1 tumor controls (4T1-TC) and SPG-56 subjects given a high dose of 240
mg/kg of body weight (4T1-SPG). Twenty days after inoculation, the mice
were injected with 10 μl/g D Luciferin, Potassium Salt via the tail vein. Ten
minutes later, the mice were anesthetized by intraperitoneal injection of 10%
chloral hydrate, and the distribution of luminescence in the mice wasscanned using a VILBER FUSION FX7 Spectra multifunctional imager(VILBER LOURMAT, France), at a wavelength of 560 nm.

Figure 1.

RESULTS
Figure 1. SPG-56 suppressed the metastasis of 4T1 cell in nude mice.
(A) 4T1 tumor control (4T1-TC) and (B) SPG-56 with high dose of 240
mg/kg body weight (4T1-SPG). 4T1-Luc breast carcinoma cells were
inoculated into nude mice, to examine whether SPG-5 inhibited theexpansion and metastasis of breast cancer in vivo. 20 days after
inoculation, the distribution of luminescence in the mice were detectedby FUSION FX7 Spectra (Vilber Lourmat) at a wavelength of 560 nm.
The color scale indicates the luminescence intensity per pixel, thehigher the luminescence intensity detected, the more 4T1-Luc tumor
cells are present. The tumor luminescence in the TC group wasdistributed not only in the tumor at site of inoculation, but also in othertissues, including the liver and lungs (Fig. 1A). This suggests thatmetastasis of the tumor occurred. However, the luminescence in thehigh dose of SPG-56 group (Fig. 1B) was greatly reduced, existing onlyin the inoculated tumor area. This indicates that SPG-56 treatment
controlled the growth of the tumor, and effectively inhibited
metastasis.

A.

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CONCLUSION
The in vivo experiment made use of in situ injection of tumor cells into
the nude mouse mammary fat pad. Compared to subcutaneous
inoculation, this inoculation method can better simulate the growth ofbreast cancer in its usual environment. These results demonstrate theabilities of the Fusion FX7 Spectra (Vilber Lourmat, France) in imaging
the effects of SPG-56 in vivo and its biodistribution pattern in thetreatment of breast cancer in mice.

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B.