Evaluating The Effects of Traditional Herbal Medicine X-911 on Cytokine Expression and Glucose Uptake in LL2 Cells With 18F-FDG/MicroPET

Traditional Chinese medicine (TCM) is empirically based, with a history of over 4000 years, and is used widely in China, Japan, Korea, Singapore, and elsewhere [1]. In TCM, the biological ingredients of herbal remedies are extracted from natural substances: plants, animal parts, shells, insects, and even stones and minerals. TCM is commonly used to nourish the blood, activate circulation, alleviate pain, regulate menstruation and attack tumor tissues, alone or in combination with anticancer drugs [2]. TCM can be used in cancer prophylaxis and/or treatment, for example, as Phyllanthus urinaria protects the liver from hepatocarcinogenesis and has shown anticancer activity [3]. Sho-saiko-to has hepatoprotective effects in various liver injury experiments, including the injury induced with CCl4 and D-galactosamine [4,5]. Paeoniae radix (PR) improves blood flow by its endothelium-dependent vasodilator action and potential antiaging and anticarcinogenesis activities [6].

chronic conditions [9]. In this study, we measured the dimensions of the tumor with physical tools and using 18 F-FDG PET, to determine the functional outcomes of the tumors. There were significant differences in the levels of TNF-α and granulocyte-macrophage colony-stimulating factor (GM-CSF), which are related to T-helper 1 (TH1) cells, which improve the balance between TH1/TH2 in tumor cells. We demonstrate that X-911 induces a type I immunological reaction that increases TH1 cell activity and reduces the tumor volume.

Cell Line and Inoculation
Lewis mouse lung carcinoma (LL2) cells were cultured in highglucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin solution.
LL2 cells (2×106) were injected subcutaneously at a single location within the right leg over 15 s, using a 30-gauge needle.

Physical Measurement of Tumor Size
After implantation for one week, the tumor volume (mm 3 ) was determined daily. The tumor size along three orthogonal diameters was measured with calipers and the corresponding tumor volume was calculated as d1× d2×d3 (d1 as longest diameter, d2 as diameter perpendicular, and d3 as the height of the tumor).

X-911 Feeding
X-911 was supplied by the ASC International Company. Extract from the plant is used as a TCM for human health. X-911 (0.25 g or 0.125 g) in sterile water was fed to the nude mice twice daily for two weeks. All nude mice were allocated to one of five groups, as shown in Table 1.
The region of interest (ROI) was manually placed over the tumor region. ROI analysis of the reconstructed images was performed with ASIPro software (Concorde Microsystems Inc.) to determine the tumor volume by microPET and 18 F-FDG uptake (%ID/g).

Cytokine Assays
The supernatants of the blood serum were collected from the mice at the end of the study for assays.

Physical Measurement of Tumor Size
The LL2 Lewis murine lung carcinoma had been implanted in the right leg of each nude mouse; the tumor volume was about 100 mm 3 and was considered ready for use in the study. As shown in

MicroPET Procedure
In the microPET imaging study, the difference between the groups on day 21 and day 28 was showed in Figure 2

Cytokine Assays
The levels of serum cytokines were analyzed using the Bio-Plex system. The serum cytokine levels in the X-911-fed mice and the control mice are shown in Table 2. Comparing these two groups, the levels of cytokines released predominantly from TH1 cells, such as IL2 and IFN-γ, did not differ significantly, and the results were similar for IL4 and IL5, although IL1β and IL10 differed between the two groups. However, TNF-a and GM-CSF were significantly higher in the X-911-fed mice than in the control mice (P = 0.03 and P = 0.01, respectively).  [11,12]. In this study, we used 18 F-FDG to measure the volumes of the tumors in vivo and determine whether they were associated with a specific tumor microenvironment, such as hypoxia or increased cellular proliferation. By measuring the tumor sizes daily along three orthogonal diameters and comparing these with 18 F-FDG data, the spatial information for tumor growth under different conditions was similar when acquired with microPET imaging and physical measurements. 18 F-FDG can detect an enhanced glycolytic reaction, which is often associated with the growth rate and malignancy potential of a tumor [13]. With microPET imaging, we identified a difference between day 21 and day 28 when the X-911 treatment was extended for a long time. We obtained excellent results with microPET, which demonstrated that X-911 caused a significant reduction in tumor growth between conditions 1 to 4 and 5 ( Figure 3) but did not affect the metabolic activity of the tumors because there was no significant difference in 18 F-FDG uptake. Therefore, X-911, a traditional Chinese medicine, can inhibit tumor growth but does not change the metabolism of the surviving cells.
A previous study showed that traditional Chinese medicines could be used in sport to relieve exhaustion, increase athletic prowess, and increase the blood vessels required to carry the nutrients essential for the rapid growth of solid tumors [14].
During tumor growth, the immune system is balanced between the Th1 (autoimmune) and Th2 (pregnancy or suppressive) responses,

Conclusions
This study has shown that X-911, a traditional Chinese medicine, can alter the balance between Th1 and Th2 cells. X-911 increased the levels of the serum cytokines released from T H 1 cells, thus inhibiting tumor cell growth. We also successfully monitored tumor formation and tumor metabolism using 18 F-FDG/microPET.
In assessing tumor development, the physical measurements were similar to those made with microPET imaging. However, only 18 F-FDG/microPET can identify the tumor metabolic status in vivo.
The glucose uptake of the surviving cells did not differ from that of normal cells. To further investigate the antitumor activity of X-911, we plan to use different kinds of tumors to compare their mechanisms.