Shikonin inhibits viability and proliferation of PCSCs
In the first part of the study, we generated PCSCs using sphere formation assay over 2–3 generations. The PCSCs in the ultra-low attachment flasks with serum-free medium formed non-adherent free-floating spheres, while differentiated adherent prostate cancer cells shrank and died [26]. Figure 1a shows the generation of the spheres within 10 days. DU145 and PC-3 and the corresponding PCSCs were treated with different concentrations of shikonin (0, 1.5, 3, 6, 12, 24, and 48 µM) for 24 h and 48 h, see Additional file 3 Fig. S1. The results of the drug sensitivity assay show that shikonin inhibited the cell viability in a dose-dependent manner (Fig. 1b). The half-maximal inhibitory concentration (IC50) analyzed by logit regression model using the SPSS software mounted around 0.75 µM for DU145, 4 µM for DU145 CSCs, 5 µM for PC-3, and 7 µM for PC-3 CSCs. Using different concentrations ( 0.5x IC50, IC50, and 2x IC50) an inhibiting effect of shikonin on the proliferation rate was also observed (Fig. 1c and Additional file 3 Fig. S1). Results showed that DU145 CSCs and PC-3 CSCs were much less sensitive to shikonin than the adherent DU145 and PC-3 cells. It supported the point that cancer stem cells were a special cell population like drug-resistant cells.
Shikonin inhibits migration and invasion of PCa cells
Since the migration and invasion ability are two important biological characteristics, we evaluated the effect of shikonin on migration and invasion of DU145, PC-3 and their corresponding sphere cells. Migration was evaluated using the scratch wound healing assay. After treatment with different concentrations of shikonin pictures were taken at different time points. Shikonin inhibited the migration of both the adherent cells and sphere cells to the center of the gap (Fig. 2a-b and Additional file 4 Fig. S2). Invasion ability was determined using the Boyden chamber system. Cells were treated again with different concentrations of shikonin (0.5 × IC50, 1 × IC50, and 2 × IC50) for 48 hours. The numbers of invaded cells were decreased significantly in the adherent PCa cell lines and even in the corresponding PCSCs, (Fig. 2c-d). It can be concluded that shikonin was able to inhibit migration and invasion remarkably in PCa cells.
Shikonin enhances the antitumor effect of cabazitaxel
To investigate if shikonin enhances the antitumor effects of cabazitaxel, we performed the viability, proliferation, and invasion assay. First the IC50 of cabazitaxel was determined. DU145 cells were treated with different concentrations of cabazitaxel (0, 1, 2, 4, and 8 nM) for 48 h. Results showed that cabazitaxel inhibited the viability of DU145 cells in a dose-dependent manner, the IC50 concentration was around 3 nM (see Additional file 5 Fig. S3). Then, DU145 and DU145-CSCs were treated with 0.75 µM shikonin combined with different concentrationsd of cabazitaxel for 48 h. Shikonin alone repeatedly resulted in a significant decrease in both viability (Fig. 3a and Additional file 6 Fig. S4) and proliferation (Fig. 3b and Additional file 6 Fig. S4) in DU145 and DU145 CSCs compared to cabazitaxel alone. The effect was more pronounced in the combination treatment, even at a lower concentration of cabazitaxel (< IC50). Similarly, the number of apoptotic cells was higher following treatment with cabazitaxel plus shikonin (Fig. 3c). Also in the invasion assay a significant higher inhibition was observed (Fig. 3d). Altogether, shikonin sensitizes the antitumor effect of cabazitaxel in both DU145 and DU145-CSCs.
Shikonin induces apoptosis through ROS-mitochondria membrane potential apoptosis pathways in PCa cell lines and PCSCs
The effect of shikonin on the apoptosis rate was also tested in the cell line PC-3 and the corresponding PC-3 CSCs. Results showed that shikonin dramatically induced apoptotic death both in the PCa cell lines DU145 and PC-3 and the corresponding PCSCs (see Additional file 7 Fig. S5).
Reactive oxygen species (ROS) generation is an essential effect of shikonin during cell death in several types of cancers [27–30]. To study the role of shikonin in prostate cancer cells we used the cell permeable reagent 2’,7’ –dichlorofluorescein diacetate (DCFDA), a fluorogenic dye that measures ROS activity within the cell. After diffusion into the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later then oxidized by ROS into 2’, 7’ –dichlorofluorescein (DCF), a highly fluorescent substance. Treatment with shikonin for 6 hours dramatically enhanced ROS generation in the PCa cell lines and the corresponding PCSCs (Fig. 4a). When the cells were pretreated with the ROS scavenger, N-acetyl-L-cysteine (NAC), at 1 mM for 4 h the effect of shikonin was significantly reduced as shown in the viability assay, which proves that shikonin induces apoptosis via ROS generation (Fig. 4b).
Then we conducted staining with tetraethylbenzimidazolylcarbocyanine iodide (JC-1) to investigate if shikonin disrupted the mitochondrial membrane potential of PCa cells and PCSCs. According to the kit instructions due to low mitochondrial membrane potential, JC-1 is predominantly a monomer that yields green fluorescence with emission of 530 ± 15 nm. At high concentrations (due to high mitochondrial membrane potential), the JC-1 aggregates
yielding a red to orange colored fluorescence (590 ± 17.5 nm) Both monomer and aggregate forms were measured, and a ratio between the two measurements was obtained and plotted. Exposure to shikonin for 48 h dramatically reduced aggregates fluorescence, indicating that shikonin induced apoptosis through ROS formation and triggered the disruption of the mitochondrial membrane potential. NAC reversed mitochondrial dysfunction and verified our conclusion (Fig. 4c). Besides this, cabazitaxel alone cannot influence ROS generation in DU145 cells (Fig. 4d). It suggested that cabazitaxel cannot interrupt the antitumor effect of shikonin.
Shikonin inhibits the expression of ABCG2 and ALDH3A1 in PCSCs.
There are different cancer stem cell markers known for prostate cancer driving drug-resistance, and cancer relapse as for example ABCG2, ALDH, CD44, CXCR4 [7]. Figure 5a and Additional file 8 Fig. S6 demonstrates that shikonin inhibited the expression of both ALDH and ABCG2 (CD338) shown by flow cytometry. Similarly, RT-qPCR showed that shikonin inhibited the expression of ALDH3A1 and ABCG2 (Fig. 5b). In some publications it was reported that increased expression of the drug transporter gene ABCG2, was detected in the ALDH + cell population [31, 32]. Inhibition of ALDH1A1 activity decreased the expression of ABCG2 and reduced chemotherapy resistance in ovarian cancer cell lines [33].To explore if shikonin downregulates the expression of ABCG2 through blocking the expression of ALDH3A1, we silenced ALDH3A1 using siRNA for 48 h and tested ABCG2 expression by RT-qPCR. The results showed that downregulation of ALDH3A1 could not influence the expression of ABCG2 (Fig. 5d). However, in the confocal fluorescence microscopy it was observed that ALDH3A1 was overlapped with ABCG2 in DU145 CSCs (Fig. 5c). Both the Pearson correlation coefficient and the overlap coefficient were 0.9 analyzed by the colocalization finder of image J software (Fig. 5d). It can be suggested that ALDH3A1 and ABCG2 were at least co-expressed in DU145 CSCs. To investigate the relation between ABCG2 and ALDH3A1, we silenced the ABCG2. After downregulating ABCG2, the expression of ALDH3A1 decreased (Fig. 5e). This implies that shikonin can block the expression of ALDH3A1 by inhibiting ABCG2.
Shikonin reverses the resistant-state of caba-DU145 through inhibiting the expression of ABCG2 and ALDH3A1
In clinical practice, cabazitaxel has proved to be effective for docetaxel-resistant CRPC patients [24]. When the CRPC patients become resistant to cabazitaxel, there are few therapeutic options left. According to our results, ABCG2 and ALDH3A1 are two primary cancer stem cell markers that can drive drug-resistance and their expression can be regulated by shikonin. Then we wanted to know if shikonin can abolish the cabazitaxel-resistant state by inhibiting the expression of ABCG2 and ALDH3A1. We generated a cabazitaxel-resistant DU145 cell line (caba-DU145) by gradually increasing the dose of cabazitaxel over eight months (see Additional file 9 Fig. S7). Then the caba-DU145 cells were pretreated with the ABCG2 inhibitor Ko143 at 1 µM. After five days, the cell viability of caba-DU145 response to cabazitaxel was determined. Results showed that after treatment with Ko143 the cells were more sensitive to cabazitaxel compared to the untreated cells (Fig. 6a). The same was observed using the proliferation assay (Fig. 6b). Also, treatment with the ALDH3A1 inhibitor CB29, showed a higher sensitivity of the cells to cabazitaxel compared to the untreated cells (Fig. 6c). The same effect was observed again using the proliferation assay (Fig. 6d). Furthermore, inhibiting ALDH3A1 and ABCG2 using CB29 and Ko143, decreased the apoptosis rate of caba-DU145 cells (Fig. 6e). As we expected, using low concentration of shikonin at 0.375 µM in combination with cabazitaxel at 3 nM increased the apoptotic rate compared to cabazitaxel alone in the caba-DU145 cells (Fig. 6f).
Our results suggested that shikonin not only enhances the antitumor effect of cabazitaxel but also reverses the cabazitaxel-resistant state by inhibiting the expression of ABCG2 and ALDH3A1, and ABCG2 suppresses the expression of ALDH3A1.