Inhibitory effects of isocryptotanshinone on gastric cancer

Gastric cancer (GC) is one of the most common digestive malignancies globally, and the prognosis of patients with advanced tumors remains poor. Isocryptotanshinone (ICTS), isolated from Salvia miltiorrhiza, was found to inhibit the proliferation of lung and breast cancer cells. However, whether ICTS has anticancer activities against GC is unknown. In the present study, we reported that the proliferation of GC cells was inhibited by ICTS in a dose- and time-dependent manner. After treatment with ICTS, GC cells were arrested in the G1/G0 phase of cell cycle and the apoptotic cells were induced in a dose-dependent manner. Additionally, ICTS suppressed the expression of cell cycle- and apoptosis-associated proteins (e.g., Cyclin D1, phosphorylated Rb, E2F1, Mcl-1, Bcl-2, and Survivin). ICTS inhibited the phosphorylation of STAT3 in a dose-dependent manner. Down-regulated STAT3 attenuated the expression of Cyclin D1, p-Rb, and Survivin, which remarkably increased the sensitivity of ICTS in GC cells; overexpression of STAT3 restored the cell growth and proliferation and the protein expression suppressed by ICTS. ICTS also suppressed the xenograft tumor growth in BALB/c nude mice. Together, these data indicate that ICTS inhibits GC proliferation by inducing G1/G0 cell cycle arrest and apoptosis via inhibiting the STAT3 signaling pathway.

chemical structure of ICTS is shown in Fig. 1A. As shown in Fig. 1B, ICTS inhibited the proliferation of SGC-7901 cells in a dose-dependent manner, and the IC 50 was 6.77 μM. As shown in Fig. 1C, after treatment with 10 μM ICTS, the proliferation of SGC-7901 cells was suppressed in a time-dependent manner. Additionally, ICTS induced inhibition of SGC-7901 cell proliferation mainly during the first 24 hours. Meanwhile, as shown in Fig. 1D and E, MKN-45 cell growth was also inhibited by ICTS in a dose-and time-dependent manner, which was consistent with the results in SGC-7901 cells, with an IC 50 of 33.1 μM. Interestingly, the inhibition of SGC-7901 cell proliferation induced by ICTS at lower concentration was more potent compared with that of MKN-45 cells. These results indicated that ICTS suppressed the proliferation of GC cells and might function as a GC suppressor.
ICTS induced GC cell cycle arrest at the G1/G0 phase. Cell proliferation is controlled by the progression of cell cycle. Here, we assessed whether ICTS inhibited cell proliferation via regulating cell cycle. As shown in Fig. 2A, treatment with ICTS for 24 hours arrested SGC-7901 cells in the G1/G0 phase of the cell cycle in a dose-dependent manner, which was consistent with the results shown in Fig. 1B. At a concentration of 10 μM, ICTS markedly increased the proportion of SGC-7901 cells in the G1/G0 phase from 47.9% to 65.7%. Meanwhile, the increase of the SGC-7901 cell proportion in the G1/G0 phase was accompanied with a concomitant decreasing proportion of cells in the S and G2/M phases of the cell cycle. Additionally, treatment with 10 μM ICTS increased the SGC-7901 cell number in the sub-G1 phase significantly, which suggested that ICTS might also play an essential role in the regulation of apoptosis. Similarly, as shown in Fig. 2B, ICTS also induced cell cycle arrest in the G1/G0 phase in MKN-45 cells and increased cell proportion in the sub-G1 phase of cell cycle at higher concentration. Together, the data showed that ICTS arrested GC cell cycle in the G1/G0 phase.

ICTS induced GC cell apoptosis.
To determine whether the ICTS-mediated growth inhibition in SGC-7901 and MKN-45 cells is associated with apoptosis, the apoptotic cells were further examined by the flow cytometry analysis. As shown in Fig. 3A and B, the percentage of Annexin + /PI + SGC-7901 cells induced by ICTS after 24 hours was significantly increased from 3.8% to 44.2% in a concentration-dependent manner. The results also indicated that even low concentration of ICTS (e.g., 2.5 and 5 μM) showed apoptosis-inducing effects and the apoptotic cell number was remarkably increased by ICTS at higher concentration (40 μM) after 24 hours. Apoptosis, which is initiated by active caspase-9, induces cleaved-PARP expression and cell death 28 . As shown in Fig. 3C, the expression of cleaved caspase-9 and PARP were significantly upregulated in SGC-7901 cells after exposed to ICTS for 24 hours. As shown in Fig. 3D and E, ICTS also promoted MKN-45 cell apoptosis in a dose-dependent manner. Additionally, ICTS treatment increased the expression of cleaved caspase-9 and PARP remarkably in MKN-45 cells (Fig. 3F). Thus, these results showed that ICTS induced apoptosis in GC cells.

ICTS regulated expression of cell cycle-and apoptosis-associated proteins in GC cells.
To further identify the molecular mechanism of GC cell growth inhibition induced by ICTS, we investigated the expression of cell cycle-and apoptosis-associated protein markers after treatment with ICTS. As shown in Fig. 4A, in SGC-7901 cells, treatment with a concentration of 20 or 40 μM ICTS significantly downregulated the phosphorylation of Rb at Ser-807/811, and the expression of Cyclin D1 and E2F1, which induces the transcription of target genes required for DNA synthesis in the late G1/S phase 29 . Meanwhile, as shown in Fig. 4B, after treatment with a high concentration of ICTS (20 and 40 μM) for 24 hours, the expression levels of Mcl-1, Bcl-2, and Survivin were also decreased significantly in SGC-7901 cells. The decrement of cell cycle-and apoptosis-associated proteins was consistent with the inhibition of cell proliferation. Similarly, as shown in Fig. 4C and D, the same changes of protein markers induced by ICTS in MKN-45 cells were also observed. These results further confirmed the cell cycle arrest and apoptosis induced by ICTS in the GC cells.
ICTS suppressed phosphorylation of STAT3. As Jak2/STAT3, MAPK, and PI3K/Akt signaling pathways played pivotal roles in the occurrence and development of GC 30 , we assessed whether ICTS had a regulatory effect on the indicated pathways. As shown in Fig. 5A, ICTS inhibited the phosphorylation of STAT3 at Tyr-705 in a dose-dependent manner and had weak effect on the total protein. It increased the phosphorylation of Akt at Ser-473 and no significant effects were observed on the phosphorylation of Erk1/2 at Thr-202/Tyr-204. However, ICTS, at a higher concentration, decreased the expression levels of Akt and Erk1/2. Interleukin (IL)−6, a pro-tumorigenic cytokine, is associated with poor survival in GC patients and Helicobacter pylori-induced STAT3 activation at Tyr-705 31,32 . As shown in Fig. 5B and C, phosphorylation of STAT3 was upregulated remarkably after stimulated by 25 ng/ml IL-6 treatment in SGC-7901 cells, and pre-treatment with ICTS significantly suppressed the IL-6-induced phosphorylation of STAT3. As shown in Fig. 5D, downregulation of STAT3 attenuated the expression of Cyclin D1, p-Rb, and Survivin, which remarkably increased the sensitivity of ICTS in SGC-7901 cells. Overexpression of STAT3 enhanced the growth of the SGC-7901 cells and the expression of Cyclin D1, p-Rb, and Survivin, which restored the cell proliferation and the protein expression suppressed by ICTS ( Fig. 5E and F). Together, ICTS inhibited GC cell growth and decreased the expression levels of cell cycle-and apoptosis-associated proteins via inhibiting the STAT3 signaling pathway.

ICTS inhibited xenograft GC growth in nude mice.
To determine the effect of ICTS on xenograft tumor growth in vivo, we established the subcutaneous SGC-7901 tumor model. The mice were randomly divided into two groups which received ICTS or vehicle injected intraperitoneally. The results showed a gradual increase in tumor volume in both groups. However, compared with the vehicle group, the tumor volume in the ICTS group increased significantly slower. On the 28 th day, the difference of tumor volume between the control and ICTS groups were significant ( Fig. 6A and B). Furthermore, phosphorylation of STAT3 in xenograft tumor was identified using immunohistochemistry assay. Results showed that phosphorylation of STAT3 in SGC-7901 tumor was suppressed by ICTS (Fig. 6C). Thus, ICTS inhibited SGC-7901 xenograft tumor growth in vivo.

Discussion
In the present study, we found that ICTS significantly inhibited the proliferation of both undifferentiated (MKN-45) and moderately differentiated (SGC-7901) GC cells in a dose-and time-dependent manner, and slowed the xenograft SGC-7901 tumor growth in mice. Cell proliferation is controlled by the activation of the checkpoints during DNA synthesis and chromosome segregation, which protects cells from the attack by genotoxic agents and leads to the inhibition of cyclin-dependent kinases (CDKs) and cell cycle arrest 33 . Three interphase CDKs (CDK2, CDK4, and CDK6), mitotic CDK1, and 4 different classes of cyclins (Cyclin A, Cyclin B, Cyclin D, and Cyclin E) are directly involved in driving the cell cycle and the aberrant expression of the CDK-cyclin complexes resulting from cancer-associated mutations induces unscheduled re-entry into the cell cycle or proliferation 34 . For instance,   Cyclin D1, Cyclin D2, and Cyclin D3 bind and activate CDK4 and CDK6 to phosphorylate the Rb protein, which leads to the subsequent release of E2F1 and the transition from G1 to S phase in the cell cycle 35 . Our present study showed that ICTS induced cell cycle arrest in the G1/G0 phase and inhibited the expression of Cyclin D1, pRb, and E2F1 in GC cells in a dose-dependent manner. As an essential protein in the G1-to-S phase transition, E2F1 binds to its dimerization partner 1 or 2 and induces the transcription of the target genes required for DNA synthesis in S phase, such as Cyclin D1, Cyclin E, CDC2, and CDC25A 36 . Park et al. 24 found that cryptotanshinone diminished the E2F1 transcriptional activity compared with the empty vector. Interestingly, this study showed that 2.5 μM ICTS suppressed the expression of E2F1 significantly compared with the 5 μM group after multiple tests. Therefore, the down-regulation of the cell cycle-associated proteins might result from the inhibition of the transcriptional activity of E2F1. Cryptotanshinone-induced cell cycle arrest in the G1/G0 or G2/M phase was dependent on the cell line type, and the effect of ICTS on cell cycle arrest might also be not the same in other cancer cells, which is deserved to be further explored 37 .
To investigate the effect of ICTS on apoptosis of GC cells, the percentage of apoptotic cells was analyzed using flow cytometry and the anti-apoptotic proteins were detected by the Western blot analysis. The results indicated that ICTS arrested GC cell cycle in the G1/G0 phase and inhibited the expression of Mcl-1, Bcl-2, and Survivin, which was in accordance with the experimental results in A549 and MCF-7 cells 26,27 . Mcl-1 and Bcl-2, the anti-apoptotic proteins of the Bcl-2 family, regulate programmed cell death via directly inhibiting the pro-apoptotic proteins 38 . Recent studies showed that cryptotanshinone induced apoptosis of pancreatic cancer 39 , prostate cancer 40 , chronic myeloid leukemia 41 , multiple myeloma 42 , glioma 43 , and lung cancer 44 cells by suppressing the anti-apoptotic proteins. Chen et al. 45 found that the cryptotanshinone-induced caspase-dependent cell death inhibited the expression of anti-apoptotic (Bcl-2 and Mcl-1) and survival proteins (Survivin) by activating the Jun N-terminal kinase (JNK) pathway in cancer cell lines. The Bcl-2 family proteins were proved to be down-regulated by nature compounds, which were already used in clinical trials 46 . Small-molecule suppressors of Survivin (e.g., YM155, Tetra-O-methyl nordihy-droguaiaretic acid, and LY2181308) were reported to show anti-cancer activities both in vitro and in vivo, and some of them have been applied in clinical anti-cancer therapy [47][48][49][50][51] . Our in vivo investigation further suggested that ICTS might be a nature compound which could be potentially applied in clinical GC treatment, and further investigations are warranted in this regard.
The persistent activation of STAT3 was found in multiple human cancers 52 , and plays a prominent role in mediating drug resistance during chemotherapy and targeted cancer therapies [53][54][55] . Researches have shown that activated STAT3 promoted the proliferation and invasion of GC cells in vitro 56,57 . Additionally, STAT3, as a prognostic marker, was associated with a poor survival in GC 58 . Serum IL-6 expression was an independent indictor for survival and a high expression level was associated with cancer development and progression in GC 32,59 . In this study, we demonstrated that ICTS attenuated the phosphorylation of STAT3 stimulated by IL-6 in GC cells, which is consistent with the results in A549 cells 26,27 . It has been found that the activation of STAT3 elevated the levels of anti-apoptotic (Mcl-1 and Survivin) and cell cycle-regulating proteins (Cyclin D1) 60,61 . When STAT3 was down-regulated by siRNA transfection, the expression of CylinD1 and Survivin was also significantly decreased (Fig. 5B). Meanwhile, upregulation of STAT3 attenuated the inhibition of Cyclin D1, p-Rb, and Survivin induced Several issues are noteworthy in this study. Firstly, the efficacy of the investigated compound is relatively low as reflected by the proliferation assay. Nevertheless, based on our data and one publication 62 , the inhibition potency of ICTS was stronger than cryptotanshinone in SGC-7901 and MKN-45 cells. Different inhibitory effects of ICTS on different gastric cancer cells could be due to their discrepant differentiation potentials and cell types, which would be explained by further related studies. Secondly, after treatment by ICTS at 10 µM, >70% of the growth-inhibited SGC-7901 cells did not undergo apoptosis, indicating that the observed cell death could be largely due to reasons other than apoptosis. We also suggested that cell cycle inhibition would be one of the mechanisms, the others of which warrant further exploration. Notably, p-AKT was increased but total AKT inhibited by ICTS in a concentration-dependent manner (Fig. 5A), and the underlying mechanism calls for further explorations.
In conclusion, the present study provided the first evidence that ICTS inhibited GC cell proliferation by inducing cell cycle arrest and apoptosis through inhibition of the STAT3 signaling.

Materials and Methods
Reagents. ICTS was obtained from ChemFaces, and the purity (98%) was verified using high performance Cell proliferation assay. The effect of isocryptotanshinone on cell proliferation was determined by CCK-8 assay. Briefly, cells were cultured in 96-well plates at a density of 2 × 10 3 cells per well and attached overnight, and they were then treated with isocryptotanshinone of different concentrations or DMSO (Beyotime) as vehicle and incubated for appropriate time. CCK-8 solution was added followed by incubation for 1 to 3 hours at 37 °C and with 5% CO 2 , and the absorbance per well was measured at 450 nm wavelength using a universal microplate reader (Bio-tek). Results were shown as the relative ratio compared with the control group which was set as 1.
Cell cycle analysis. Cells were seed in 6-well plates at a density of 1 × Cell apoptosis detection. Cells were seeded into 6-well plates at a density of 1 × 10 5 cells per well and allowed to grow overnight. After treated with isocryptotanshinone (2.5-40 μM) or DMSO for 24 hours, cells were harvested and washed twice using cold PBS, and re-suspended in Annexin-binding buffer containing PI and annexin-v in dark at room temperature for 15 minutes. The stained cells were subjected to FACScan flow cytometry (Becton-Dickson Biosciences) using the CellQuest software, and the percentage of apoptotic cells was analyzed using the Flow Jo software.
Western Blot analysis. After treatment with isocryptotanshinone or DMSO, cells were lysed in RIPA lysis buffer containing 1 mM PMSF (Beyotime). The concentration of protein samples was detected using the BCA Protein assay kit (Beyotime). About 30 micrograms of the total protein were separated using 10% SDS-PAGE and transferred to a PVDF membrane. After blocked in TBS-T (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% Tween) containing 5% non-fat milk for 90 minutes, the membranes were incubated with primary antibodies at 4 °C overnight. The membranes were then washed with TBS-T, and exposed to HRP-conjugated goat-anti-rabbit/ mouse secondary antibodies at room temperature for 60 minutes. Blots were detected by chemiluminescence using Super Signal West Femo (Thermo Scientific) and bands were captured using a digital imaging system (Tanon).
Scientific RepoRts | (2018) 8:9307 | DOI:10.1038/s41598-018-27638-0 Animal experiment. Ten 4-week-old male BALB/c nude mice (weight, 20 ± 4 g) were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. All mice were maintained under specific pathogen free conditions at 22 °C with 55% humidity and a 12-h light/12-h dark cycle. 10 6 SGC-7901 cells were subcutaneously injected into the upper flank region of the nude mice. After 2 weeks, all mice were divided into two (ICTS and control) groups which were intraperitoneally injected with 20 mg/kg ICTS or vehicle every other day for a total of 4 weeks. Tumor sizes of nude mice were measured every 4 days. Tumor volumes (V) were calculated following V = π/6 × a × b 2 . At the end of the experiments, mice were subsequently sacrificed by cervical dislocation after anesthesia.
Then the SGC-7901 tumors were harvested and fixed in 4% poly-formal-dehyde for 2 days. We detected the phosphorylation of STAT3 in tumors by immunohistochemistry staining as previously described 15,64 . The primary antibody replaced by PBS was regarded as the negative control and the known positive tissue section staining was considered as the positive control.
Statistical analysis. The quantitative data obtained from at least triplicate wells were presented as mean ± SE. The non-linear regression analysis was performed using the GraphPad Prism software to calculate the IC 50 . The qualitative differences between 2 groups were analyzed using the Student's t test. P values of <0.05 and <0.01 were considered statistically significant and very significant, respectively.
Ethics statement. The in vivo part of the current study was performed according to the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Care and Use Committee of Anhui Medical University.