Dihydrotanshinone I inhibits ovarian cancer cell proliferation and migration by transcriptional repression of PIK3CA gene

Abstract Dihydrotanshinone I (DHTS), extracted from Salvia miltiorrhiza, was found to be the most effective compound of tanshen extracts against cancer cells in our previous studies. However, the therapeutic benefits and underlying mechanisms of DHTS on ovarian cancer remain uncertain. In this study, we demonstrated the cytocidal effects of DHTS on chemosensitive ovarian cancer cells with or without platinum‐based chemotherapy. DHTS was able to inhibit proliferation and migration of ovarian cancer cells in vitro and in vivo through modulation of the PI3K/AKT signalling pathways. Combinatorial treatment of DHTS and cisplatin exhibited enhanced DNA damage in ovarian cancer cells. Overall, these findings suggest that DHTS induces ovarian cancer cells death via induction of DNA damage and inhibits ovarian cancer cell proliferation and migration.

lipophilic abietane diterpene compounds extracted from the dried root of Salvia miltiorrhiza. Tanshinone IIA, cryptotanshinone, tanshinone I and dihydrotanshinone I (DHTS) are four major constituents of tanshinones. 13 Recently, several studies have shown that tanshinone I, dihydrotanshinone I and tanshinone IIA could exert pro-apoptotic and cytotoxic effects on a number of human cancer cell lines 14,15 and also inhibit epithelial-mesenchymal transition (EMT) and migration. 16,17 Induction of apoptosis is well-accepted as one of the most promising therapeutic strategies for cancer treatment. 18,19 Previous studies revealed the pro-apoptotic ability of DHTS in human erythroleukemia, glioma, osteosarcoma and colorectal cancer in both in vitro and in vivo settings. [20][21][22][23][24][25] In addition, emerging evidence suggests that DHTS treatment attenuated cell migration by down-regulating adhesion molecules VCAM-1 and ICAM-1 in osteosarcoma cells. 22 Research on DHTS has revealed its beneficial effects with regard to apoptosis induction and migration suppression. However, the therapeutic benefits and underlying mechanisms of DHTS on ovarian cancer remain uncertain.
In this study, we evaluated the therapeutic efficacy of DHTS

| Cell culture
Human ovarian cancer cell lines, A2780 and OV2008, were purchased from American Type Culture Collection (ATCC). Normal ovarian epithelial cell line IOSE80 was obtained from the Canadian Ovarian Tissue Bank. Cells were maintained in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin (streptomycin 100 μg/mL, penicillin 100 U/mL) at 37°C with 5% CO2 in a humidified incubator.

| Cell viability assay
The effect of DHTS on the viability of cells was detected by MTT assay. Cells were seeded into 96-well plates (~1 × 10 4 /well) and treated with various concentrations of DHTS (0,1, 2, 4, 6, 8 and 10 μM). At the indicated time-points, the old medium was replaced with 100 μL of fresh medium containing 20 μL of MTT solution (5 mg/ mL in PBS). After 4 hours of incubation at 37°C, formazan crystals were solubilized with 150 μL DMSO in each well. The absorbance levels for each sample at 540 nm were measured using a Multiskan Ascent plate reader (Thermo Electron, New York, NY, USA). The data were duplicated five times at least.

| Wound healing assay
Wound healing assay was performed to evaluate the migration of ovarian cancer cells. Briefly, cells were seeded into 24-well plates and grown until confluent state and then were scratched by using P1000 sterile tips. The plates were rinsed twice with sterile PBS to remove detached cancers cells. Fresh complete cell culture medium was added with various concentrations of DHTS. Cell migration was observed under a phase-contrast microscope at 100 × magnification at 0 and 24 h post-scratch. Migrated cells in the denuded area in each of six random fields were measured and quantified with a computerassisted microscope.

| Transwell migration and invasion assay
Cell migration and invasion were evaluated by the transwell assay.
For the transwell migration assay, ovarian cancer cells treated with DHTS were seeded into the upper chambers with FBS-free medium at a density of 5 × 10 4 cells per well and 600 μL of complete growth medium with 10% FBS was placed in the lower chamber as a chemoattractant. For the transwell invasion assay, 1 × 10 5 cells per chamber were plated. Cells were allowed to invade through the Matrigel-coated inserts. After 24 hours incubation at 37°C, noninvading cells remaining on the upper side of the chamber were removed with cotton swabs. Cancer cells that on the underside of the filter were stained with 0.1% crystal violet (Sigma) for 10 minutes and counted under a microscope at 200 × magnification. Six random fields of each transwell membrane were counted and averaged.

| Microarray expression analysis
Ovarian cancer cells were treated with either DHTS or vehicle for 24 h and harvested for total RNA extraction by using TrizolTM reagent (Life Sciences). The RNA concentration and quality were evaluated by NanoDrop spectrophotometer (Thermo) and denaturing agarose gel(1.5%) electrophoresis. Cancer cell cDNA was synthe-

| Colony formation assay
Cells were seeded in six-well plates at a density of 500 cells per well. Then, medium with DHTS or vehicle was changed every 3 days for ~ 15 days when most of the colony contained more than 50 cells.
After cold PBS washing, the colonies were fixed using 70% ethanol and stained with 0.1% crystal violet dissolved in 10% ethanol at room temperature. Then, the counts of cell colonies were manually scored, and the images were recorded under a computer-assisted microscope.

| Western blot
Total protein was isolated from the cells using RIPA lysis buffer  After injection, the fish embryos were immediately transferred into housing-keeping water. Injected embryos were kept at 28°C and were examined every other day for monitoring tumour growth and invasion using a fluorescent microscope.

| Statistical analysis
Data are presented as means ± standard error of the mean (SEM) for three independent experiments. Statistical differences between different groups were analysed by using Student's t test by GraphPad Prism 5.0 (San Diego, CA, USA). A significant difference was considered at P < .05.

| DHTS inhibits migration and invasion of ovarian cancer cells
Women with malignant or even low malignant ovarian cancer are at a very high risk of metastasis, which involves cell migration and invasion during the process and can be fatal to patients. 22 (Figure 2A, B, right panel, P < .01). Consistent with the wound healing assay, DHTS also significantly inhibited ovarian cancer cell migration and invasion, as determined by transwell chamber assays, in a dose-dependent manner (Figure 2A, B, lower panel). Treatment with 0.5-2 μM DHTS inhibited migration by 39%-68% in A2780 cells, and 25%-61% in OV2008 cells, as compared to controls, respectively.
Similarly, cancer cell invasion was suppressed by 23%-81% in A2780 and 41%-72% in OV2008 (Figure 2A, B, right panel). These results suggested that DHTS can suppress the migratory and invasive abilities of ovarian cancer cells in a concentration-dependent manner.  Figure 3B). Among these targeting genes, we found that PIK3CA, DDB2 and HGF mRNA expression were significantly down-regulated in A2780 cells exposed to DHTS ( Figure 3C). In addition, Western blotting analysis showed that PI3Kα expression and its downstream protein AKT phosphorylation in A2780 treated with DHTS were also significantly down-regulated ( Figure 3D). These data implied a potential role of DHTS in the regulation of PIK3CA gene transcription in ovarian cancer cells.

| DHTS suppressed proliferation of ovarian cancer cells by transcriptionally regulating endogenous PIK3CA gene expression
Preclinical investigations have suggested that the PI3K/AKT pathway is frequently activated in ovarian cancer. [32][33][34] Thus, this pathway is regarded as an attractive candidate for cancer therapeutic interventions.  Figure 4A). Meanwhile, cell proliferation assay showed that A2780 cells stably expressing exogenous PIK3CA developed resistance to DHTS induced anti-proliferation ( Figure 4B).
Additionally, clone formation assay showed that the clone-forming ability of stable exogenous PIK3CA expressing-A2780 cells was significantly elevated as compared to control groups in the presence of 2 μM DHTS ( Figure 4C). These data supported that DHTS treatment significantly down-regulated the PIK3CA transcription and protein levels, and followed activity of PI3K/AKT signalling pathway, leading to anti-proliferation of ovarian cancer cells.

| Overexpression of PIK3CA gene in A2780 ovarian cancer cells confers resistance to DHTSprohibited cell migration and invasion
Next, we investigated whether DHTS inhibits ovarian cancer cell migration and invasion by regulating PIK3CA transcription. Here, we used wound healing and transwell chamber assays on wild-type, empty

| DHTS sensitizes ovarian cancer cells to platinum therapy
The standard therapy of clinical management of advanced ovarian cancer following debulking surgery is chemotherapy with a or both for 24 hours, followed by immunofluorescence staining.
Interestingly, DNA double-strand break marker, formation of γH2AX foci was highly induced in combination-treated cells as compared to single agent or DMSO-treated cells ( Figure 6B). Our data suggested that DHTS significantly increased the sensitivity of ovarian cancer cells to cisplatin through the induction of DNA damage.

| DHTS inhibits ovarian cancer cell proliferation and metastasis in zebrafish ovarian cancer model
To These data demonstrated that DHTS can significantly in vivo inhibited primary ovarian cancer growth and followed tumour metastasis.

| D ISCUSS I ON
Ovarian cancer is the leading cause of death from all gynecologic cancers. 1 Currently, treatment for ovarian cancer involves platinum-based chemotherapy that has many limitations, such as rapid clearance, severe side effects, unfavourable biodistribution and drug resistance. 37,38 There is an urgent unmet clinical need in developing new therapies for patients with ovarian cancer. In the past decade, the advent of immunotherapeutic and molecularly targeted agents has dramatically revolutionized the therapeutic landscape for cancer treatment. For example, PARP inhibitor olaparib showed a 70% reduction in the risk of progression or death for ovarian cancer patients with BRCA1/2 mutation. 39 Rucaparib, a novel inhibitor of PARP, demonstrated a 56% response rate in patients with highgrade, recurrent, platinum-sensitive ovarian carcinoma and is evaluated in a phase 3 trial. 40 Our previous study has shown the promise of combining inhibitors of PI3K and PARP as treatment for ovarian cancer. 41  Currently, accumulating evidence suggests that phyto-active compounds hold promise as adjuvants of traditional chemotherapy and may be helpful for the chemo-prevention of ovarian cancer. 44,45 In our study, we found that suppression of PI3K pathway activity

| CON CLUS IONS
In this study, our study has demonstrated that targeting PI3K/AKT signalling pathway activity using DHTS in ovarian cancer represents a promising therapeutic strategy. We suggest the administration of DHTS, as an adjuvant for chemotherapeutic agents, for ovarian cancer patients with high PI3K activation to overcome ovarian cancer invasion and metastasis.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.