Chinese Formula Ganji Fang Inhibits Tumor Growth Through Regulating Cell Mitosis by Reducing ARPP- 19 Expression in Hepatocellular Carcinoma

Chunlei Zhang Shanghai University of Traditional Chinese Medicine Kaiyue Tang Shanghai University of Traditonal Chinese Medicine Lili Yang Shanghai University of Traditional Chinese Medicine Yang Liu Shanghai University of Traditional Chinese Medicine Yifan Zhang Shanghai University of Traditional Chinese Medicine Jinzu Yang Shanghai University of Traditional Chinese Medicine Peiyong Zheng Shanghai University of Traditional Chinese Medicine Haiyan Song (  songhy@126.com ) Shanghai University of Traditional Chinese Medicine https://orcid.org/0000-0003-2155-8110

of chemotherapy with Adriamycin, 5-uorouracil, interferon α and platinumbased combinations was con rmed to be approximately 20%. The survival bene t of chemotherapy was not proved [9].
Additionally, targeted drug and systemic chemotherapy often have some side-effects, affecting the life quality of patients [10,11].
Traditional Chinese medicine (TCM) has been used to treat malignant tumor for hundreds of years.
Recent studies have indicated that TCM can induce cell-cycle arrest and attenuate the tumor-associated macrophage-stimulated proliferation to inhibit tumor proliferation [12][13][14]. In addition, more and more researches con rmed the synergism and detoxi cation of combination of TCM with chemo-or radiotherapy [15]. Yu et al. approved that bu-zhong-yi-qi-tang in combination with cisplatin can effectively reverse cisplatin resistance via inducing apoptosis and autophagy in lung cancer cells [16]. Xiao-chai-hutang could inhibit cell proliferation and induce apoptosis of HCC cells by regulating the expression of Bax, Bcl-2, CDK4 and cyclin-D1 [17]. Studies have shown that Songyou Yin (SYY) combined with oxaliplatin can inhibit epithelial-mesenchymal transition (EMT) so that decrease the metastatic potential of the residual HCC after oxaliplatin treatment [18]. Further study showed SYY could regulate EMT through down-regulation of TGF-β1 expression and inhibition of the SMAD2/3 signaling pathway [19]. The compound PHY906 is the extract of the classical TCM formula Huang-qin-tang (HQT). It composed of Scutellaria baicalensis Georgi, Paeonia lacti ora Pall, Glycyrrhiza uralensis Fisch and Ziziphus jujuba Mill [15]. PHY906 combined with some anticancer drugs have synergistic effects in cancer therapy. The toxicity and side effect of chemotherapy could be signi cantly alleviated by PHY906 treatment [20]. It can be used for the adjuvant of antineoplastics (Sorafenib, CTP-11, 5-FU, leucovorin (LV) and capecitabine) in the treatment of advanced colorectal, pancreatic, and HCC [21,22].
Ganji Fang (GJF) is an empirical formula used for tumor treatment in Longhua hospital for decades.
Clinical investigations have shown that GJF could relieve tumor growth, improve symptoms of HCC patients and extend the life span of advanced HCC patients. One year survival rate of patients with advanced HCC with GJF treatment was improved (GJF group=31%, control group=4.5%) [23]. The subcutaneous tumor growth of colon adenocarcinoma could be attenuated by GJF through inhibiting p53 mutations [24]. However, the potential anti-cancer mechanism of GJF is not yet clari ed.
ARPP-19 (cAMP-regulated phosphoprotein 19) is a member of the alpha-endosul ne (ENSA) family [25]. It plays a role in tumorigenesis and development through regulating cell proliferation, cell cycle and survival [26][27][28][29]. It has been shown that the PP2A is a crucial downstream signaling molecule of ARPP-19 and suppress the growth of tumor [26]. Other research shows that ARPP-19 could regulate the pathogenesis of breast and prostate cancer by vasoactive intestinal peptide (VIP) [27,28]. Dopamine phosphorylates and induces expression of ARPP-19 [27], which plays an indispensable part in the initiation and progression of cancer [29]. Our previous studies demonstrated that elevated ARPP-19 level in HCC tissues and downregulation of ARPP-19 decreased the activation of mitotic substrates, resulting in cell cycle arrest at the G2/M phase in various human HCC cells [30]. These results indicate that ARPP-19 is a potential target of anti-cancer drugs. Therefore, in this study, we investigate the effect of GJF on HCC in vivo with HCC xenograft mice model and try to explore its potential mechanism associated with inhibition of tumor proliferation through regulating ARPP-19.  15, 9, 9, 12, 9, 15, 15, 15, 15, 3, 9, 9, 9, 9, 6, 6 and  HepG2 cells were digested and washed with ice-cold PBS. Subsequently, 5×10 6 HepG2 cells in 200 uL DMEM were injected into the right axillary region of the BALB/c-nude mice. Afterwards, mice were randomly assigned into three groups (n = 8 per group). The mice of GJF-L group and GJF-H group received intragastric administration of low or high dose of GJF once a day, respectively (The low dose is calculated according to the standard dose of clinical practice for adult and the equivalent dose of adult and mouse body surface area, and the high dose is twice of the low dose). The control group was administrated with normal saline by gavage. The treatments lasted for 4 weeks. The activity of xenograft mice was observed and body weight was measured every four or ve days. The length and width of tumors was detected with a vernier caliper every four or ve days. The volume of tumor was calculated depending on the following formulas: volume= a * b 2 /2, "a" is the maximal width and "b" is the maximal orthogonal width [31]. On the 28th day of treatment, all mice were weighed and sacri ced. Then the tumors were excised and weighed. Mice livers and kidneys were removed, then frozen or xed in 10%
For the investigation of survival time, 1×10 7 SMMC 7721 cells were injected into the right axillary region of the BALB/c-nude mice. The tumor-bearing mice were allocated into two groups (Control and GJF-H, n=8 per group) and treated as above description. The death number of mice was recorded in either natural death or euthanasia due to the large tumor (diameter >15 mm), and the survival time was analyzed.

Immunohistochemistry
For the immunohistochemistry analysis of Ki-67 and ARPP-19, tumor tissues were embedded in para n and 4-μm thick sections were sliced. Tissue sections were depara nized, rehydrated, and subjected to antigen retrieval by immersing sections in citrate buffer (10mM; pH 6.

Histological examination of liver and kidney Tissues
The liver and kidney sections were stained with H&E according to the standard methods. In brief, the fresh tissue samples were xed in 10% formalin and were embedded in para n. The samples were then crosscut into slices of 4 µm and were stained with H&E staining solution (Kehui). Subsequently, the stained sections were observed and photographed under a light microscope (with magni cation of 200×).

Western Blot
Page 6/20 The tissues were lysed with RIPA lysis buffer with protease and phosphatase inhibitors for 30min on ice. Then, the tissues were centrifuged at 12,000 rpm for 15 min at 4°C.

Statistical Analysis
In this experiment, the technical statisticians conducted statistical analysis by Statistical Product and Service Solution (SPSS) statistics sofware (IBM, New York, USA). The data were expressed as mean ± standard deviation (SD). Statistical analyses were carried out using one-way ANOVA, followed by Tukey's post-hoc test to assess the differences between the two groups. The two-tailed Student's t-test was used for the analysis between two groups. p<0.05 is regarded statistically signi cant.

Results
GJF improved general health state of the HCC tumorbearing mice and exhibited no toxic effects After intragastric administration of GJF for 28 days, compared with model group, favorable effects in the general status, such as more food intake and better mobility were observed in HepG2-xenografted nude mice with GJF treatment. Moreover, as shown in Fig.1, GJF increased the body weight of nude mice in a dose-dependent manner. Furthermore, GJF caused no toxic pathological changes in the tissue structure of liver and kidney in HepG2-xenografted nude mice (Fig.2).
GJF treatment suppressed tumor growth of HCC tumorbearing nude mice We evaluated the anti-cancer effect of GJF on male nude mice with HepG2 xenograft tumor. After 28 days administration of GJF, the volume of tumors was shown to be lowered in GJF formula-treated groups when compared with untreated control group. The effect of high dose is more signi cant (p < 0.01). The tumor weights of mice after treatments with high dose of GJF formula were signi cantly decreased than those of untreated control mice (p < 0.05), and low dose of GJF had the tendency to decrease tumor weight (Fig. 3).

GJF inhibited cell proliferation of HCC tumor
The Ki67 protein is a cellular marker for cell proliferation and tumor growth. The Ki67 was detected within the cell nucleus by IHC. Compared with the control group, tumor tissue of GJF-H or GJF-L groups showed less Ki67 staining. The positive expression rate of Ki67 is also called Ki67 proliferation index (PI), which is normally incorporated in the evaluating indexes for malignant degree of tumor. Fig.3b showed GJF reduced the Ki67 PI signi cantly in a dose-dependent manner.
Proliferating cell nuclear antigen (PCNA) is also closely related to DNA synthesis and can be used as an indicator of cell proliferation. The protein level of PCNA was lowered in the tumor treated by GJF obviously (Figure 3c, 3d).

GJF down-regulated ARPP-19 expression of HCC tumor tissues
The ARPP-19 plays an important role in HCC cell proliferation. Previous research has shown that the major characteristic of ARPP-19 was that ARPP-19 took effect on HCC pathogenesis through decreasing protein levels of phospho-(Ser) CDKs substrates and increasing levels of inactivated cyclin division [30].
To investigate whether GJF could impact the ARPP-19 expression, it was measured by western blot and IHC in the tumor tissues of HepG2-xenografted nude mice. As shown in Figure 4(a) and Figure 4(b), all treatments with GJF inhibited the protein levels of ARPP-19 compared with the control group (P < 0.01).
Moreover, the effect of GJF depends on the given doses.

GJF inhibited the activation of mitosis-related protein of HCC tumor tissues
Some studies have shown that a subfamily of cyclin-dependent kinases (Cdks) promote the mammalian cell cycle. Cyclins consists of complicated combinations of Cdks and speci c regulatory proteins. Then, Cdk-Cyclin drive the cell forward through the cell cycle [32]. Mitotic substrates measure the cell division. Cdc2 phosphorylate mitotic substrates which regulate the G2/M transition directly. Therefore, we detect the effect of GJF on the level of phosphorylation of Cdc2 and Cdks substrates in the tumor tissues of HepG2-xenografted nude mice by using an antibody recognizing the phospho-serine Cdk consensus motif. As shown in Figure 4(c), all treatments with GJF reduced the protein expression of phosphorylation of mitotic substrates (p<0.05). A signi cantly elevated level of inactivated Cdc2 (phospho-cdc2 (Tyr15)) was also found in GJF groups (p< 0.05), indicating that GJF could attenuate the activation of Cdc2.
Herbal formula treatment had the tendency of prolonging survival of HCC tumor-bearing mice The survival time of mice in the treatment groups were recorded. As shown in Table 1, the mice treated with GJF-H lived for longer period. After tumor cell inoculation, the median survival days of mice in GJF-H group was 41.6 days, which was a bit longer than that in control (37.25 days) group. Although there is no statistical difference, which might be due to less animal number and the limitation of tumor volume leading to short observing period, there is the tendency.  Table 1 The survival time of HCC tumor-bearing mice Discussion TCM offers potential for HCC treatment with the fewer adverse effects. In order to guarantee the validity and reliability, evidence-based preclinical and clinical explorations are required. Moreover, elucidation of the underlying pharmaceutical mechanism could bene t the acceptance of TCM application [12][13][14][33][34][35].
In the past 50 years, the Department of Oncology, Longhua Hospital, Shanghai University of Traditional Chinese medicine has been devoted to the clinical practice and mechanism exploration of the prevention and treatment of digestive tumors by TCM. According to the TCM pathogenesis theory of liver cancer, "spleen de ciency is the internal basic, qi stagnation, blood stasis, damp heat, toxin and phlegm turbidity are the manifestation characteristics" and clinical experiences, under the leadership of Professor Qiu Jiaxin, the anti-tumor formula "Ganji Fang" has been developed, with the function of "invigorating the spleen and regulating qi, clearing away heat and detoxi cation, softening and resolving phlegm". Clinical application has shown that GJF can prolong the survival time of patients with advanced liver cancer.
Early animal experiments showed that GJF can prevent the occurrence of HCC induced by diethylnitrosamine (DEN) [36]. GJF has cytotoxic effect on HCC cells, and the whole formula is the strongest. Some ingredients, such as atractylodes macrocephala, oyster and rhizoma arisaematis, have anti-mutagenic effect, while atractylodes macrocephala and poria cocos make cells disable to start malignant transformation [23]. In this study, we also nd the tumor-inhibiting effect of GJF. The subcutaneous HCC tumor volume and weight was obviously down-regulated by GJF treatment dosedependently. Moreover, the xenografted nude mice with GJF treatment had improved food intake, activity and body weight. And the prolonged survival tendency was observed in mice of GJF group. These results indicate the effect of GJF on tumor growth and life quality. In addition, no toxic pathological changes in the tissue of liver and kidney suggested safety of application.
To further explore the anti-tumor mechanism of GJF, Ki67 proliferation index (PI) and PCNA, the proliferation indictor closely related to tumor malignancy were determined. The results showed GJF signi cantly reduced the Ki67 PI and PCNA level in a dose-dependent manner. The cell cycle is crucial in tumor proliferation and growth. Our previous researches con rmed that ARPP-19 participated in tumor growth of HCC [30]. The molecule plays an important role in the pathogenesis of HCC by regulating cell cycle [37][38][39][40]. Therefore, we measured the expression level of ARPP-19 of the HCC tumor tissues. The IHC and western blot results showed obvious decreased ARPP-19 in GJF group than control dosedependently.
Previous studies have shown the function of ARPP-19 in cell mitosis [38]. Cyclin B-Cdc2 is the universal regulator of mitosis phase of cell cycle. Activated Cyclin B-Cdc2 phosphorylates mitotic CDK substrates to trigger the mitosis. ARPP-19 could be activated by a starter amount of activated Cdc2 through a kinase greatwall (Gwl) or activated directly by Cdc2. In turn, it plays a role in the activation and maintenance of cyclin B-Cdc2 activity [38,40]. Additionally, it can also inhibit dephosphorylating mitotic substrates during M phase. Therefore, ARPP-19 is involved in the core element of the autoregulatory loop of cyclin B-Cdc2 [39,41]. In consistence with ARPP-19 expression, we also observed higher level of inactivated Cdc2 (phospho-cdc2) and lower Cdks mitosis substrates in tumor tissues of GJF group than control group. Therefore, the regulation of cell cycle through ARPP-19 is closely associated with the effect of GJF (Figure 7).

Conclusion
In summary, this study shows the effect of GJF in inhibiting tumor growth of HCC without toxicity, and can improve the quality of life of tumor bearing nude mice. The anti-tumor effect of GJF is related to the inhibition of HCC cell proliferation by arresting cell mitosis through down-regulating ARPP-19. This study provides a scienti c basis for the treatment of HCC with TCM. However, the follow-up random clinical control trials are still need to be carried out to con rm the e cacy. In addition, since the multi-target effect of traditional Chinese medicine, the relevant mechanism research also needs to be further explored in order to promote its application in HCC treatment.  Figure 1 GJF increased the body weight of HCC tumor-bearing nude mice. Data represented mean ± SD, n = 8, ** p < 0.01 vs. control.  b Comparison of the Ki67 proliferation index (PI) of three groups, n = 8. c The PCNA protein levels of tumor tissues analyzed by western blot, with β-actin serving as an internal reference. d Quanti cation of data in c, n = 4. All data were shown as mean ±SD, * p < 0.05, *** p < 0.001 vs. control.  GJF regulated activation cyclin-dependent kinases and phosphorylated mitotic substrates. a

Figures
Representative blot image of Cdks substrates and Cdc2 protein expression and activation of tumor tissues analyzed by Western blot, with β-actin serving as an internal reference. b Quanti cation of data in b analyzed by using densitometry. Data represented mean ± SD, n = 4, * p < 0.05 and ** p < 0.01 vs. control.

Figure 7
Sketch of the effect of GJF on tumor cell proliferation through regulating ARPP-19 promoted cell mitosis. Activation of cyclin B-Cdc2 is important to trigger cell mitosis. The small starter amount of active cyclin B-Cdc2 could activate ARPP-19, which in turn helps maintain activation of cyclin B-Cdc2. In addition, ARPP-19 plays a role in preventing inactivation of the substrates of Cdc2. Gangji Fang could downregulate ARPP-19 to arrest cell mitosis, thereby inhibit cell proliferation.