Jujuboside B Reverse CUMS-Promoted Tumor Progression via Blocking PI3K/Akt and MAPK/ERK and Dephosphorylating CREB Signaling

Background Jujuboside B (JUB) is a saponins isolated from the seeds of Zizyphi jujuba var. spinosi, which is used to treat mental illness and is reported recently to induce cancer cell apoptosis. As our previous research showed chronic stress promoted tumor growth, this work aims to investigate whether JUB exert antitumor effect in addition to its antidepressant effect and possible mechanism. Methods 56 female C57BL/6 mice were grouped into 7 groups: A (blank control), B (tumor-bearing control), C (tumor-bearing + JUB), D (CUMS control), E (CUMS + JUB), F (tumor-bearing + CUMS), and G (tumor-bearing + CUMS + JUB). Groups C, E, G, B, D, and F were administered, respectively, with JUB (40 mg/kg/day) or vehicle for 2 weeks. Serum 5-HT, Trp (tryptophane), inflammatory cytokines TNF-α, IL-4, -6, and -10 levels were detected by ELISA. The tumors in groups B and F were isolated for RNA-seq sequencing. Protein and mRNA expression of Bax, Bcl-2, p-PI3K, p-Akt, p-MAPK, p-ERK, and p-CREB in tumor tissues were detected. In vitro, A549 cells were stimulated with JUB (60 μmol/L), in which proliferation rate and colony formation rate were detected. The PI3K/Akt and, MAPK/ERK pathway were measured. Results Chronic stress successfully induced the depression-like phenotype (group D vs. A) and promoted tumor growth (group B vs. F). JUB significantly ameliorated the depression-like phenotype and increased 5-HT, Trp levels (group D vs. E), and reversing CUMS-induced tumor progression. Meanwhile, JUB decreased inflammatory cytokine levels. Chronic stress upregulated the phosphorylation levels of PI3K/Akt/MAPK/ERK/CREB; JUB reversed this regulation. JUB significantly inhibited cell viability, colony formation rate, and downregulated the phosphorylation levels of PI3K/Akt/MAPK/ERK/CREB in vitro. Conclusions JUB reverses CUMS-promoted tumor progression in tumor-bearing mice with depression-like phenotype. JUB exerts the dual beneficial effect on tumor growth and depression-like phenotype by blocking the signal transduction pathway of PI3K/Akt, MAPK/ERK, and dephosphorylating the downstream signaling regulator CREB.


Introduction
Jujuboside B (JUB) is a saponin isolated from the seeds of Zizyphi jujuba var. spinosi, which is used to treat mental illness, neurodegenerative diseases, and so on. It is reported to possess multiple pharmacological activities. Recently, JUB is found to induce cancer cell apoptosis and exert anticancer activity in breast and colon cancer [1,2].
Depression is a common comorbidity in cancer patients. In our previous work, we noted that about 30% of cancer patients occurred mild to major depression [3,4]. Many factors lead to comorbid depression in cancer patients. First of all, being confirmed as malignancy is a great negative life event for most people. Second, the side effects and toxicities of chemotherapy/radiotherapy are often severe and intolerable for most cancer patients, and the prognosis is usually poor. Finally, cancer treatment costs are giant, and it is an unaffordable economic burden for ordinary people [5,6]. Depression comorbidity reduces treatment compliance, weakens therapy outcomes, and increases cancer mortality. Therefore, antidepressant treatment is of importance and necessary for the cancer patient's comorbidity with depression [7] .
To make it worse, chronic stress might promote tumor growth. Our previous research showed that chronic stress promoted tumor growth in the mice model. [8,9] As JUB has a variety of pharmacological activities, this work aims to investigate whether JUB exerts an antitumor effects in addition to its antidepressant effects, and a possible mechanisms based on tumor-bearing mice with depression-like phenotype induced by chronic unpredictable mild stress (CUMS).

Open-Field Test (OFT).
The OFT focused on observing the animals' locomotion and exploratory behaviors. The mice should be familiarized with the testing environment for at least 3 h. Animals were placed into the box (50 × 50 × 40 cm) in the same position in turn, and the animal behavior analysis software was opened to spontaneously record the activities of the mice for 5 min. After each mouse was tested, 75% alcohol solution was used to avoid odor interference.

Sucrose Preference Test (SPT)
. Followed by 12 h of 2% sucrose solution for adaption, then 18 h of water deprivation, mice were exposed to 2% sucrose solution and water for 2 h. The intake of liquid was recorded and calculated.
2.6. Animal Grouping. Fifty-six female mice were randomly divided into 7 groups (n = 8): A (blank control), B (tumorbearing control), C (tumor-bearing + JUB), D (CUMS control), E (CUMS + JUB), F (tumor-bearing + CUMS), and G (tumor-bearing + CUMS + JUB). Seven groups of mice were given, respectively, different manipulations ( Figure 1). Mice in CUMS-induced groups were fed separately and given nine different stressors randomly for 8 weeks (Table 1). To avoid prediction, the stressors were not repeated consecutively. 8 weeks later, to verify the CUMS model established successfully (groups D, E, F, and G), LLC cells (2 × 10 5 ) were inoculated subcutaneously into the right flank of the animals (groups B, C, F, and G). After 3 days of cancer cell inoculation, JUB (40 mg/kg/day) was administrated intraperitoneally to mice (groups C, E, and G) for 2 weeks. Simultaneously, mice in group A, group B, group D, and group F were given vehicle intraperitoneally. Finally, all animals were executed, and serum and tumors were collected. Tumor weight and volume were measured.
2.7. RNA Sequencing. The total RNA of tumor tissue was isolated using the Trizol reagent following the protocol. Generated the cluster and sequenced libraries, 150 bp paired-end reads were collected. About 63 G reads and 430 M clean reads for all samples were collected. The clean read was calculated using cufflinks, and the read counts were obtained by HTSeq-count. P 3 Journal of Immunology Research < 0:05 and fold change >2 or <0.5 were considered statistically significant.
2.9. mRNA Expression Study. Total RNA from tumor tissue was isolated with Trizol following the standard procedure. For RT-qPCR, RNA reverse transcription and PrimeScript RT-qPCR kit were performed according to the protocol (Takara, 9109, RR037A). The 7500 Real time Quantitative PCR systems were used to run PCR. Primer sequences were synthesized by Sangon Biotech (Table 2).

Protein Expression Analysis.
Tumor tissue proteins were obtained with RIPA lysate (NCM Biotechnology, China). Then, the level of protein was detected by the BCA method (Yoche Biotechnology, China). Proteins were sequentially denatured, electrophoresed, and transferred to an NC membrane (Merk  2.11. Statistical Analysis. All values were expressed as means ± SD. Student's t-test was used in comparison between two groups, and one-way analysis of variance (ANOVA) (two-tailed) was used in the inter group. P value <0.05 was considered statistically significant.

Behavioral Establishment of CUMS Model.
After 8 weeks of model establishment, there was a striking difference in the behavioral and sucrose preference test between CUMS model mice and normal feeding mice. Compared with baseline, the behavioral scores (including locomotion score and exploratory score) and pleasure scores (sucrose preference) were decreased in CUMS group. Interestingly, there was no change in normal feeding animals ( Figure 2). These data suggested that chronic stress successfully induced a depression-like phenotype.

JUB Ameliorated the Depression-Like Phenotype.
In nontumor groups, the behavioral scores (including locomotion score and exploratory score), pleasure scores (sucrose preference), 5-HT, and Trp levels of mice in group E (CUMS + JUB) were increased compared with those in group D (CUMS control) after 2 weeks with JUB administration. Meanwhile, in the tumor group, the depression-like behaviors of mice in group G (tumor-bearing + CUMS + JUB) were ameliorated compared with mice in group F (tumor-bearing + CUMS) ( Figure 3). Thus, JUB could ameliorate depression-like phenotype in tumor and nontumor groups.  (Figures 4(a) and 4(b)). In addition, CUMS and JUB did not affect the body weight in all groups (Figure 4(c)).
3.5. CUMS Activated CREB; JUB Dephosphorylated the Downstream Signaling Regulator CREB. CREB is a protein involved in emotions, tumor cell apoptosis, and immune response. In the JUB group, p-CREB-1 was noticeably decreased in comparison with that in mice of group B (tumor-bearing control) and group F (tumor-bearing + CUMS), respectively. The protein and mRNA levels of proapoptotic Bax in mice of group C (tumor-bearing + JUB) and in mice of group G (tumor-bearing + CUMS + JUB) were significantly increased, while the protein and mRNA levels of antiapoptotic Bcl-2 was significantly decreased in comparison with group B (tumor-bearing control) and group F (tumor-bearing + CUMS). These results displayed that chronic stress enhanced the Bcl-2, p-CREB expression, and downregulated Bax expression ( Figure 6); however, JUB administration reversed this phenomenon.
3.6. JUB Blocked the Signal Transduction Pathway of PI3K/Akt and MAPK/ERK. Based on the existing literature, PI3K/Akt and MAPK/ERK are two crucial upstream cascades of activated CREB. In our study, CUMS activated the pathway of PI3K/Akt and MAPK/ERK. In group F (tumor-bearing + CUMS), PI3K-Akt and MAPK single pathways were activated compared with the tumor control group (Figure 7(a)). The p-PI3K, p-Akt, p-MAPK, and p-ERK protein expression in JUB groups was significantly decreased in comparison with the corresponding control group. Thus, CUMS enhanced the signal transduction pathway of PI3K/Akt and MAPK/ERK, but JUB blocked these signal transductions.

JUB Inhibits Tumor Progression In Vitro.
The half maximal inhibitory concentration (IC50) of JUB was about 60 μmol/L in A549 cells (Figure 8(a)). JUB reduced clonal formation in comparison with the control group (Figure 8(b)). Moreover, JUB blocked the signal transduction pathway of PI3K/Akt and MAPK/ERK, dephosphorylating the level of PI3K, Akt, MAPK, and CREB (Figures 8(c)-8(h)). The results in vivo were consistent with in vitro.  Journal of Immunology Research

Discussion
For a decade, chronic stress or chronic stress-induced depression-like phenotype has been documented to promote cancer progression [10][11][12]. Chronic stress induces lowgrade inflammation and impaired immune homeostasis [13][14][15], and finally promoted tumor growth [16,17]. Clinical studies also revealed that chronic depression increased cancer occurrence and contributed to the development of malignant tumors [18]. Cancer patients with major depression were more possible correlated with cancer metastasis [10,11,19]. Generally, stressful life experiences are related to poorer cancer prognosis, lower survival, and higher mortality. Therefore, it is important to pay attention to depression comorbidities in cancer treatment.
Much evidence demonstrated that some antidepressants exerted antitumor effects [20][21][22]. For example, fluoxetine has been found to play a proactive role in antitumor progression in lung cancer and hepatocellular carcinoma [21]. Sertraline was able to regulate cancer multidrug resistance [20]. In our previous studies, we found that fluoxetine reverses multidrug resistance in breast cancer cells which might be medicated by inhibition of glutathione s-transferase-π and p-glycoprotein [4,12]. And lately, we confirmed that the antidepressant drug fluoxetine exerts an antitumor effect via inhibiting enzymes related to the knurine pathway and enhancing T cellular immunity in NSCLC animals [8].
As traditional Chinese medicines usually have multiple pharmacological activities, in this work, we focused on Jujuboside B, the most effective component of the dried seed of Zizyphi jujuba var. spinosi (Bunge) Hu ex H.F. Chou [23]. We established the chronic stressed tumor-bearing comorbidity mice model successfully as confirmed by the behavioral scores (locomotion and exploratory scores) and sucrose preference test. The model was consistent with the core symptoms of anhedonia and social activity decline in patients with depression.
Our results suggested that chronic stress has a detrimental impact on oncotherapy, and JUB might have some reverse effects. On the one hand, the tumor in CUMS group mice was bigger than the tumor in non-CUMS group. JUB remarkably reversed the tumor-promoted compared with vehicle, in addition to its depression-like phenotype amelioration in chronic stress mice. Our work was consistent with 7 Journal of Immunology Research research that JUB exerted anticancer effects in acute leukemia, gastric cancer, and colon cancer [1,2,15]. On the other hand, chronic stress or depression and inflammation usually fuel one another [24]. Chronic stress has a detrimental impact on immune system functions both in human beings and in animals [25]. In this study, chronic stress increased TNF-α and IL-4, -6, and -10 levels. JUB significantly reversed these upregulations, which implied that the antitu-mor effect of JUB may be related to the stress-immunecancer axis.

Conclusions
This study reveals that depression can promote the development of cancer; JUB exerts dual antidepressant and antitumor effects in tumor-depression comorbidity model mice. The antitumor effect of JUB on depression and tumor progression by blocking the signal transduction pathway of PI3K/Akt and MAPK/ERK and dephosphorylating the downstream signaling regulator CREB is shown in Figure 9. These findings also reminded us to pay attention to the treatment of cancer patients with depression and provide new perspectives on the molecular targets of JUB.

Data Availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.