Apoptotic effect and cell arrest of deoxyshikonin in human osteosarcoma cells through the p38 pathway

Osteosarcoma is the most common primary bone cancer that affects adolescents with early metastatic potential and drastically reduces their long‐term survival rate if pulmonary metastases are detected at diagnosis. The natural naphthoquinol compound deoxyshikonin exhibits anticancer properties, so we hypothesized that it has an apoptotic effect on osteosarcoma U2OS and HOS cells and studied its mechanisms. After deoxyshikonin treatment, dose‐dependent decreases in cell viability, induction of cell apoptosis and arrest in the sub‐G1 phase of U2OS and HOS cells were observed. The increases in cleaved caspase 3 expression and the decreases in X‐chromosome‐linked IAP (XIAP) and cellular inhibitors of apoptosis 1 (cIAP‐1) expressions after deoxyshikonin treatment in the human apoptosis array were identified in HOS cells, and dose‐dependent expression changes of IAPs and cleaved caspase 3, 8 and 9 were verified by Western blotting in U2OS and HOS cells. Phosphorylation of extracellular signal‐regulated protein kinases (ERK)1/2, c‐Jun N‐terminal kinases (JNK)1/2 and p38 expressions in U2OS and HOS cells was also increased by deoxyshikonin in a dose‐dependent manner. Subsequently, cotreatment with inhibitors of ERK (U0126), JNK (JNK‐IN‐8) and p38 (SB203580) was performed to show that p38 signalling is responsible for deoxyshikonin‐induced apoptosis in U2OS and HOS cells, but not via the ERK and JNK pathways. These discoveries demonstrate that deoxyshikonin may be a possible chemotherapeutic candidate to induce cell arrest and apoptosis by activating extrinsic and intrinsic pathways through p38 for human osteosarcoma.

CT of the metastatic sites of the skeletal bones should be routinely surveyed for early detection of metastases and reduction of cancer-associated deaths during osteosarcoma diagnosis. 4 According to the radiological staging of osteosarcoma, the combination of neoadjuvant chemotherapy and limb preservation surgery has increased long-term survival rates to approximately 68%. [5][6][7] However, early lung metastases are responsible for one of the most lethal paediatric malignancies, 8 so novel agents that target particular intracellular signalling pathways related to antiosteosarcoma cells are in urgent need of development.
Apoptosis, a process of programmed cell death, is hallmarked by distinctive morphological and biochemical characteristics that regulate physiological growth and tissue homeostasis. 9,10 By intracellular signalling pathways such as mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, apoptosis is modulated to promote healthy cell division, turnover, immunity and accurate development and cell death. [11][12][13] In addition to initiator caspases 8 and 9 and effector caspases 3 and 7, several modulators of apoptosis, such as X-chromosome-linked IAP (XIAP) and cellular inhibitors of apoptosis 1 and 2 (cIAP-1 and 2), are involved in the apoptotic pathway. [14][15][16] Therefore, understanding the stress-inducible molecules that trigger apoptosis in response to chemotherapy for human osteosarcoma attracts researchers to develop molecular-targeted therapy through extrinsic and/or intrinsic pathways. Currently, most anti-osteosarcoma strategies in clinical therapy focus on inducing apoptosis of cancer cells, whether they metastasize or not.

| Annexin V-FITC apoptosis staining assay
After treatment with the experimental concentration range of deoxyshikonin, about 8.5 × 10 5 U2OS and 6.5 × 10 5 HOS cells were cultured in a 6 cm plate for 24 h. Subsequently, U2OS and HOS cells were harvested by trypsinization together with floating nonviable cells. We used the FITC Annexin V Apoptosis Detection Kit I based on the manufacturer's protocols (BD Biosciences) to determine the apoptotic effect of deoxyshikonin on U2OS and HOS cells, and also performed flow cytometry to analyse their cell cycles. 29 In combination with annexin V-FITC apoptosis staining and PI staining were measured to differentiate apoptosis from necrosis.

| Human apoptosis array
To explore the underlying mechanism of induced apoptosis, we used the Human Apoptosis Array Kit to examine protein lysates from cells treated with vehicle or 20 μM deoxyshikonin-treated cells for 24 h. According to the manufacturer's protocols (R&D Systems), the kit was used to detect 35 human apoptosis-related proteins simultaneously. The captured proteins were detected with biotinylated detection antibodies and deposited on the nitrocellulose membrane and visualized with chemiluminescent detection reagents. 30

| Preparation of cell lysates and Western blot analysis
To verify the results of the human apoptosis array analysis and to explore the underlying signalling pathways of the molecular mechanism, Western blot analysis was performed. As previously described, we seeded 8.5 × 10 5 U2OS and 6.5 × 10 5 HOS cells in 6-cm dishes for 16 h and examined them in the experimental deoxyshikonin concentration range for 24 h, and prepared total cell lysates of U2OS and HOS cells. [31][32][33] We performed Western blot analysis using specific primary antibodies against the initiator and effector caspases 8, 9 and 3, cleaved caspases 8, 9 and 3 and specific antibodies against the unphosphorylated or phosphorylated forms of the three corresponding MAPKs (ERK1/2, JNK1/2 and p38) to identify possible pathways.
Then we incubated the blots with horseradish peroxidase goat antirabbit or anti-mouse IgG for 1 h and measured the intensity of each band using densitometry.

| Statistical analysis
One-way analysis of variance (anova) with post hoc Tukey test were performed for more than two groups with equal sample sizes per group, respectively. Repeating each experiment at least three times (n ≥ 3) was performed independently. If p values <0.05, it was considered statistically significant.

| Cytotoxicity of deoxyshikonin in U2OS and HOS cells
To assess deoxyshikonin ( Figure

| Deoxyshikonin activates extrinsic and intrinsic apoptotic pathways in U2OS and HOS cells
To identify the underlying mechanism, we performed the human apoptosis array to examine proteins related to deoxyshikonin

F I G U R E 3 Analysis of deoxyshikonin on cell apoptosis in U2OS and HOS cells. (A) U2OS
and HOS were treated with deoxyshikonin (0, 2.5, 5, 10 and 20 μM) for 24 h and then subjected to flow cytometry after PI and Annexin V-FITC/PI staining to analyse DNA contents. The cell cycle profile of flow cytometry was subsequently quantified. Cells that were considered viable were FITC Annexin V and PI negative; cells that were in early apoptosis were FITC Annexin V positive and PI negative; and cells that were in late apoptosis or already dead were both FITC Annexin V and PI positive. (B) Thereupon quantitative analysis of early apoptosis and late apoptosis was summed up to differentiate apoptosis from necrosis. intrinsic caspase 9 mediated cascades and converging on their downstream effector caspase 3.

| Deoxyshikonin activates extrinsic and intrinsic apoptotic pathways through p38 signalling in U2OS and HOS cells
MAPKs may form part of the signalling pathways that directly affect caspases 8, 9 and 3 to regulate apoptosis. As shown in Figure 6A  herbs are increasingly used as adjuvant cancer treatments for relative safety, poor solubility in water, low bioavailability and drug resistance reduce the efficacy of additional chemotherapy, such as curcumin and shikonin. [34][35][36][37][38] Therefore, their various analogues have been developed for cancer cells that increase cytotoxicity, inhibit proliferation, improve sensitized chemotherapy and inhibit metastases. 25,39,40 In addition to multidrug resistance protein 1 and breast cancer resistance protein (BCRP)1, the new shikonin analog deoxyshikonin bypasses drug resistance through the drug trans-  ERK and JNK inhibitors; that is, deoxyshikonin induces apoptosis in U2OS and HOS cells through the p38 pathway. Surely, our results suggest that deoxyshikonin may be a potential anticancer candidate to induce apoptosis of human osteosarcoma. However, the promising anti-osteosarcoma mechanism of deoxyshikonin needs to be further studied for the successful treatment of human osteosarcoma, and further pharmacological and clinical investigations are required in vivo. writing -original draft (equal); writing -review and editing (equal).

This study was supported by Chung Shan Medical University
Hospital, Taiwan (CSH-2023-D-008).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there is no conflict of interest.

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