Propofol Regulates ER Stress to Inhibit Tumour Growth and Sensitize Osteosarcoma to Doxorubicin

Osteosarcoma is the most common malignant bone tumour affecting children and young adults. The antitumour role of propofol, a widely used intravenous sedative-hypnotic agent, has been recently reported in different cancer types. In this study, we aimed to assess the role of propofol on osteosarcoma and explore the possible mechanisms. Propofol of increasing concentrations (2.5, 5, 10, and 20 μg/ml) was used to treat the MG63 and 143B cells for 72 hours, and the CCK8 assay was applied to evaluate the tumour cell proliferation. Tumour cell migration and invasion were assessed with the transwell assay. The tumour cells were also treated with doxorubicin single agent or in combination with propofol to explore their synergic role. Differential expressed genes after propofol treatment were obtained and functionally assessed with bioinformatic tools. Expression of ER stress markers CHOP, p-eIF2α, and XBP1s was evaluated to validate the activation of ER stress response with western blot and qRT-PCR. The statistical analyses were performed with R v4.2.1. Propofol treatment led to significant growth inhibition in MG63 and 143B cells in a dose-dependent manner (p < 0.05). Osteosarcoma migration (MG63 91.4 (82–102) vs. 56.8 (49–65), p < 0.05; 143B 96.6 (77–104) vs. 45.4 (28–54), p < 0.05) and invasion (MG63 68.6 (61–80) vs. 32 (25–39), p < 0.05; 143B 90.6 (72–100) vs. 39.2 (26–55), p < 0.05) were reduced after propofol treatment. Doxorubicin sensitivity was increased after propofol treatment compared with the control group (p < 0.05). Bioinformatic analysis showed significant functional enrichment in ER stress response after propofol treatment. Upregulation of CHOP, p-eIF2α, and XBP1s was detected in MG63 and 143B secondary to propofol treatment. In conclusion, we found that propofol treatment suppressed osteosarcoma proliferation and invasion and had a synergic role with doxorubicin by inducing ER stress. Our findings provided a novel option in osteosarcoma therapy.


Introduction
Osteosarcoma is a highly malignant bone tumour afecting the extremities of children and young adults. Limb salvage surgery and anthracycline (such as doxorubicin)-based chemotherapy are the frst-line treatment for osteosarcoma [1]. Despite recent developments in surgery [2,3] and systemic treatment methods [4,5], local recurrence and drug resistance still pose as signifcant challenges and lead to decreased overall survival. Tus, novel methods to deal with osteosarcoma progression and drug resistance are pressingly needed.
Propofol is a widely used intravenous sedative-hypnotic agent, and its antitumour role has recently been recognized. Te use of propofol as general anaesthesia induction in gastric cancer showed signifcantly better survival compared with using etomidate in specifc TNM stages [6]. In vivo studies with a colorectal cancer xenograft model of mice also showed a signifcant decrease in tumour development with propofol in comparison with sevofurane under nonsurgical conditions [7]. Besides tumour development, propofol also has been proposed to reverse drug resistance of multiple chemotherapy agents such as cisplatin [8][9][10], docetaxel [11], 5-fuorouracil [12], and others [13] in multiple cancer types. Molecular mechanisms regarding the role of propofol on cancer have been proposed. For example, propofol has been reported to suppress lung cancer tumorigenesis by modulating the circ-ERBB2/miR-7-5p/FOXM1 axis [14]. Other studies showed that propofol suppressed colorectal cancer development by the circ-PABPN1/miR-638/SRSF1 axis [15] and mediated pancreatic cancer cell activity through the repression of ADAM8 via SP1 [16,17]. Te current understanding of possible molecular mechanisms has been extensively reviewed [18,19].
Endoplasmic reticulum (ER) is involved in the biosynthesis of lipids and proteins, and many factors or drugs could lead to "ER stress," a state induced by the accumulation of misfolded and/or unfolded proteins [20]. ER stressinduced osteosarcoma cell death was extensively reported [21][22][23][24][25][26].
In this study, we aimed to assess the role of propofol on osteosarcoma and its sensitivity to doxorubicin and explore the possible mechanism. We performed experiments on the MG63 and 143B cell lines and found that propofol inhibited cell proliferation, migration, invasion, and sensitized tumour cells to doxorubicin therapy. Our data also proved the presence of ER stress and activation of UPR pathways under propofol treatment. Based on our fndings, we proposed that propofol could inhibit osteosarcoma malignancy and promote sensitivity to doxorubicin via inducing ER stress. Tokyo, Japan). Te following primers were used for qRT-PCR: XBP1, forward: 5'-GGAGTTAAGACAGCGCTTGG-3', reverse: 5'-GCACCTGCTGCGGACTC-3'; GAPDH, forward: 5'-ACCACAGTCCATGCCATC-3', reverse: 5'-TCCACCCTGTTGCTG-3'. Gene expression was analysed using the 2 −ΔΔCt method.

CCK8
Assay. Cells in their logarithmic growth phase were seeded into a 96-well plate at a density of 1 × 10 4 cells/ well and incubated with the treatment. After 72 h, 10 μl of CCK8 solution (Shanghai, China) was added to each well and incubated for another 4 hours at 37°C. Te absorbance of each well was measured at 450 nm with a microplate reader. Each experiment was conducted with 5 biological repeats.

Transwell Assay.
Briefy, cells were suspended in the serum-free medium and seeded in the chambers of 24-well plates with or without Matrigel precoating. At 48 h culture, the chambers were taken out and penetrating cells were fxed with 5% paraformaldehyde for 20 min and dyed with 0.1% crystal violet for 20 min. Penetrating cells in 5 randomly selected felds of each sample were captured for counting using a light microscope (magnifcation 20x).

Bioinformatic and Statistical
Analysis. All statistical analyses were performed in R v4.2.1 (https://www.R-project. org/). Data were presented as mean ± SD. Te t-test was used for analysing measurement data attributed to the normal distribution and homogeneity of variance. p < 0.05 indicated the signifcant diference.

Propofol Treatment Led to Growth Inhibition in
Osteosarcoma. To assess whether propofol could afect osteosarcoma cell growth, the CCK8 assay was performed after propofol treatment. Increasing concentrations of propofol (2.5, 5, 10, and 20 μg/ml) together with DMSO control were used to treat osteosarcoma cell lines MG63 and 143B for 72 hours. Te remaining viable cells were assessed with the CCK8 assay. Te reading of OD450 showed a gradual decrease with increasing concentrations of propofol in both cell lines (Figures 1(a)  . Tose data showed that growth inhibition was observed in both cell lines in a dosedependent manner. Since propofol (2.5 μg/ml) showed a signifcant reduction in cell proliferation compared with the control group, it was used as the working concentration for the following experiments. Tese data showed that propofol treatment inhibited osteosarcoma cell migration and invasion.

Propofol Sensitized Doxorubicin-Induced Growth
Inhibition in Osteosarcoma. Next, we aimed to explore whether propofol could afect the antitumour efciency of doxorubicin in osteosarcoma. An increasing dose of doxorubicin were used to treat osteosarcoma cell lines MG63 and 143B with or without propofol (2.5 μg/ml). Te growth curves were plotted based on the OD450 readings, and the IC50s were calculated ( Figure 3). Te IC50 (μM) of doxorubicin in combination with the propofol group was signifcantly lower than that of the doxorubicin single agent treatment group (0.008 vs 0.052, p < 0.05) in the MG63. Similar changes were also observed in 143B cells. Te IC50 (μM) was reduced from 0.021 in the doxorubicin single agent treatment group to 0.014 in the doxorubicin + propofol treatment group (p < 0.05). Based on these fndings, we concluded that propofol could sensitize doxorubicininduced growth inhibition in osteosarcoma.

Propofol-Induced ER Stress in Tumour Models.
In order to explore the molecular changes after propofol treatment, we analysed the transcriptional changes after propofol treatment in the dataset GSE101724. Raw data were downloaded from the GEO database, and the expression matrix was constructed. Diferentially expressed genes were analysed between the propofol treatment group and the control group and plotted as a volcano plot (Figure 4(a)).
Signifcantly diferentially expressed genes were defned as | logFC| > 1 and p < 0.05. Altogether 195 genes met the criteria and were collected. Gene Ontology (GO) enrichment analysis on the diferential expressed genes was performed, and the top 8 activities are plotted as shown in the bar graph (Figure 4(b)). Te top 10 up-or downregulated genes based on logFC changes were plotted as a heatmap (Figure 4(c)). Interestingly, we found that most of the diferential expressed genes were enriched in endoplasmic reticulum (ER) stress-related activities. ER stress is known to be involved in multiple tumour activities such as proliferation, invasion, and drug resistance. Tese data showed a correlation between propofol treatment with ER stress, which might explain our fndings on the role of propofol in osteosarcoma in this study.

ER Stress Response Was Activated after Propofol Treatment in Osteosarcoma.
Next, we set out to test whether ER stress was involved in propofol treatment in osteosarcoma. We examined the expression of ER stress-related markers such as CHOP, p-eIF2α, and XBP1s. Western blot results showed that CHOP and p-eIF2α were upregulated after propofol treatment in both MG63 and 143B cells ( Figure 5(a)). Te presence of XBP1s was also detected in both cell lines after propofol treatment ( Figure 5(b)). All these data suggested the concerted activation of all three ER stress sensors and their combinatorial response, validating that ER stress was induced by propofol treatment.

Discussion
In this study, we examined the antitumour role of propofol in osteosarcoma and explored the possible mechanism. We frst tested the growth inhibition induced by propofol. CCK8 assay after propofol treatment showed signifcant growth inhibition in a dose-dependent manner in multiple  osteosarcoma in vitro models. Tumour migration and invasion changes after propofol treatment were also assessed with the transwell assay. Results showed that propofol treatment induced decreased cell migration and invasion in both MG63 and 143B cell lines.
Next, we tried to test whether propofol could sensitize osteosarcoma to doxorubicin treatment. Proliferation assays of osteosarcoma cells cultured with diferent concentrations of doxorubicin with or without propofol were performed. Proliferation curves were plotted, and the IC50s under diferent conditions had been calculated to refect the drug sensitivity. In accordance with similar experiments in other tumour types, propofol signifcantly reduced the IC50 and sensitized osteosarcoma to doxorubicin treatment.
We explored the possible mechanisms by evaluating the transcriptional changes after propofol treatment with bioinformatic tools. Diferentially expressed genes were collected and functional enrichment highlighted ER stress response-related pathways. We were aware that the GSE101724 study was diferent from our study in tumour cell lines and propofol concentrations. GSE101724 study was used as pilot study to predict the possible roles of propofol on cells. Te fndings were validated in our study with osteosarcoma cell lines before jumping into any conclusion.
To validate the presence of ER stress and activation of unfolded protein response (UPR) after propofol treatment in osteosarcoma, all three pathways [20] were examined by testing the protein level of CHOP and p-eIF2α together   propofol induces ER stress and autophagy by promoting calcium release and ROS production in C2C12 myoblast cell line [27]. Similar results were also observed in HeLa cells [28].
Limitations of this paper should be noticed. Tis study focused on the role of propofol on osteosarcoma and proposed the involvement of ER stress response; further studies are needed to fully illustrate the mechanisms on the detailed process of propofol-induced ER stress response. In addition, the antitumour role of propofol was based on the in vitro  TRIB3  CEBPB  SCG2  DDIT4  GDF15  SLC3A2  DDIT3  VGF  PLAU  TNFRSF12A  ROMO1  C20orf52  ID3  LOC401115  ATP5I  HMGCR  INSIG1  LOC100130516 NDUFA3 LOC100131801 (c) Figure 4: Bioinformatic analysis showed that propofol treatment led to endoplasmic reticulum stress. Transcriptional data of GSE101724 were downloaded from the GEO database and diferentially expressed genes after propofol (300 μM) treatment were functionally analysed.
Diferentially expressed genes were shown in the volcano plot (a) with up-and downregulated genes labelled as red or blue, respectively. GO functional enrichment of the diferential expressed genes showed enrichment in the endoplasmic reticulum stress-related pathways (b). Te top 10 up-or downregulated genes after propofol treatment were plotted as the heatmap (c). results of tumour cells treated with 2.5 μg/ml of propofol for a duration of 48 and 72 hours. More studies should be conducted before we could fnd the appropriate usage of propofol in clinic as an antitumour agent.

Conclusions
In conclusion, we assessed the antitumour role of propofol on osteosarcoma and found that propofol could induce the ER stress response, inhibit osteosarcoma cell proliferation, migration, and invasion, and increase the sensitivity to doxorubicin treatment. Our fndings provided a novel option in osteosarcoma therapy.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.