The effect of propofol on chemosensitivity of paclitaxel in cervical cancer cells

Abstract Background Propofol is a drug with potential anticancer effect. This study aimed to explore the effect of propofol on chemosensitivity of cervical cancer cells to paclitaxel. Methods HeLa and CaSki cells were selected for drug experiments. Cell viability was evaluated via CCK‐8 assay, and the combination index (CI) was calculated by CompuSyn software. A clinically relevant concentration and IC30 of propofol were selected in combination with 5 nM paclitaxel. BrdU incorporation, transwell, and flow cytometry assays were utilized to evaluate cell proliferation, migration, invasion, and apoptosis. The expression of β‐tubulin, stathmin 1, and GAPDH proteins was evaluated by Western blot. The stathmin 1 cDNA plasmid was used to establish stathmin 1‐overexpressing CaSki cells. Results At clinically relevant concentrations (0–80 μM), propofol did not affect cancer cell viability, but high concentrations (100–800 μM) reduced cell viability. The CI values of propofol with IC30 (200 μM in HeLa; 400 μM in CaSki) combined with 5 nM paclitaxel were <1. The effect of propofol with IC30 combined with paclitaxel on cell proliferation, migration, invasion, and apoptosis were stronger than individual effect, while 30 μM propofol had no effect. The Western blot results showed 30 μM propofol did not affect β‐tubulin and stathmin 1 expression in cells, although paclitaxel upregulated β‐tubulin expression while downregulating stathmin 1 expression. Compared with paclitaxel alone, cotreatment with propofol at its IC30 and paclitaxel decreased stathmin 1 expression but had no effect on β‐tubulin expression. High stathmin 1 expression weakened the effect of paclitaxel on cell viability and apoptosis, while propofol partially reversed these effect. Conclusion Propofol at clinically relevant concentrations had no effect on the malignant biological behaviors of cervical cancer cells, while propofol at high concentrations decreased.Propofol with IC30 and paclitaxel had synergetic effect on cancer cells through a reduction in stathmin 1 expression.


| INTRODUCTION
Propofol (2,6-diisopropylphenol), a popular intravenous anesthetic, is distinguished by its stable induction of anesthesia and rapid reversal and is widely applied in resection surgery for malignant tumors 1 and in various outpatient applications to allow painless operations. Propofolinduced anesthesia usually occurs at a blood concentration of 4-6 mg/L, which is equivalent to 22-34 μM. 2 Accumulating research has demonstrated that propofol can suppress the malignant biological behaviors of various cancer cells via many signaling pathways. 3,4 In addition, numerous studies have demonstrated that propofol reduces the resistance of cancer cells to many chemotherapeutic drugs. 5,6 For instance, propofol could increase the cytotoxicity of cisplatin in liver cancer. 5 It has been suggested that propofol might enhance the effect of chemotherapeutic drugs on tumors.
Cervical cancer is a kind of cancer that is a serious threat to the lives of women. Although HPV vaccination reduces the incidence of cervical cancer, there were still an estimated 4290 deaths from cervical cancer in the United States in 2021. 7 Surgery is the standard therapy for cervical cancer patients with early-stage disease, while chemotherapy is used in patients with postoperative recurrence and in postoperative treatment of patients with advanced disease. [8][9][10] Paclitaxel, a common anticancer drug, is a firstline chemotherapy for advanced, metastatic, and recurrent cervical cancer. 11 However, the emerging phenomenon of paclitaxel resistance might contribute to the poor prognosis of patients. 12 A study showed that propofol enhanced the lethal effect of paclitaxel on prostate cancer cells. 6 Studies concentrating on the influence of propofol on the cytotoxicity of paclitaxel in cervical cancer remain rare. Therefore, our research aimed to explore the effect of propofol at clinically relevant concentrations on the malignant behaviors of cervical cancer cells. Then, we analyzed the effect and potential mechanism of the combination of propofol at its IC30 and paclitaxel on the viability, proliferation, metastasis, and apoptosis of cervical cancer cells to assess the influence of propofol on the cytotoxicity of paclitaxel.

| Cell culture
HeLa and CaSki cell lines were provided by Shanghai Cell Biology Medical Research Institute. HeLa cells were cultured in DMEM (Gibco, USA), and CaSki cells were cultured in RPMI-1640 medium (Gibco, USA). Both media were supplemented with 10% FBS (Gibco, USA) and 1% penicillin-streptomycin antibiotic solution. HeLa and CaSki cell lines were incubated in a humidified environment of 5% CO 2 /95% air at 37°C.

| Cell counting kit-8 (CCK-8) assay
Cells (4000/well) were dispensed into 96-well plates. After cell attachment, the cells were treated with the above two series of propofol concentrations for 24 or 48 h. For paclitaxel treatment, cells were cultured with a series of concentrations of paclitaxel (0, 2.5, 5, 10 nM) for 48 h. Afterward, 10 μL of CCK-8 solution was added to each well and incubated for 2 h, and cell viability was assessed by measuring the OD 450 values with a microplate reader (Thermo, USA). Cell viability was quantified via the listed equation: Absorbance control group − Absorbance blank group . Then, the IC30 of propofol in HeLa and CaSki cells was selected for use in combination with the above concentrations of paclitaxel to evaluate the interaction of the two drugs. The experiment was repeated three times.

| Drug combination effect assay
The potential synergistic or antagonistic effect of propofol and paclitaxel was analyzed by the Chou-Talalay method. 13 The values of the combination index (CI) were computed via CompuSyn software. The relationship between the CI value and drug-drug interaction effect is as follows: CI <0.90, synergism; CI = 0.9 ~ 1.1, additive; CI >1.1, antagonism. The CI values were conducive to determine the final concentration of paclitaxel in further experiments. The subsequent proliferation and apoptosis assays were divided into two parts. In one part, the effect of 30 μM propofol on paclitaxel efficacy was explored, and in the other part, the effect of propofol at its IC30 on paclitaxel efficacy was studied. For each part, cells were divided into four groups: negative control group, propofol group, paclitaxel group, and cotreatment group.

| Bromodeoxyuridine (BrdU) incorporation assay
Cells (1.5 × 10 5 cells/well) were dispensed in 6-well plates and cultured with different treatments based on the above groups for 48 h. Then, BrdU (50 μg/mL) was added to the medium. After 6 h, the medium was removed, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. DNA was denatured with 2 mol/L HCl for 5 min, the cell membrane was permeabilized by incubation with 0.2% Triton X-100 for 20 min, and the cells were treated with 3% BSA for 1 h. The cells were rinsed three times with PBS between each step. The cells were incubated with an α-BrdU antibody (mouse mAb, CST, 1:200) at 4°C overnight. Then, the cells were rewarmed at 37°C for 30 min and washed with PBS. The cells were incubated with the secondary antibody for 30 min and rinsed three times with PBS. The cells were stained with DAB and hematoxylin and then counted under a microscope (DMi8, Leica). The experiment was performed three times.

| Flow cytometry assay
Cells (4 × 10 5 cells/dish) were seeded in 60-mm diameter dishes and incubated overnight for adherence. The cells were subjected to the above treatments for 48 h. Afterward, the cells were dissociated with 0.25% trypsin, washed with PBS and resuspended in 100 μL of 1 × binding buffer. The cells were stained with Annexin V-FITC and propidium iodide (PI) solution for 15 min in the dark. Next, 400 μL of binding buffer was added to each group, and apoptosis was evaluated by a CytoFLEX flow cytometer (Beckman Coulter, USA). The assay was carried out three times.

| Transwell assays
A total of 2 × 10 5 cells/well were dispensed in 6-well plates and incubated for attachment. the corresponding treatments were applied for 48 h, 2 × 10 4 (for the migration assay) or 5 × 10 4 (for the invasion assay) cells were collected in 100 μL of serum-free medium and then added into the top chambers (Costar, USA), which contained a membrane with or without a coating of Matrigel basement membrane matrix (Biosciences, USA). A total of 700 μL of complete culture medium was added to the lower chambers. After 24 h, the cells were treated with 4% fixative, stained with 0.25% crystal violet (Beyotime, China) and observed under a microscope. The experiment was performed three times.

| Western blot analysis
After the drug treatments were applied for 48 h, total cellular protein was extracted. The following protocol was described in our previous study. 14 The primary antibodies were a rabbit anti-stathmin 1 antibody, rabbit anti-βtubulin antibody, and rabbit anti-GAPDH antibody. The chemiluminescent bands were detected and visualized by a Bio-Rad ChemiDocTM XRS system (Bio-Rad, USA). The experiment was repeated three times.

| Cell transfection
The stathmin 1 cDNA sequence was purchased from Changsha Youbio Biosciences Inc. and was inserted into the pCDH-GFP + Puro vector to establish stathmin 1-overexpressing CaSki cells. The lentivirus particles were collected after transfection of the corresponding vectors along with the auxiliary plasmids pMD2.G and psPAX2 into HEK293T cells. Then, CaSki cells were infected with the lentiviruses, and 2 μg/mL puromycin was utilized to select cells with stable stathmin 1 overexpression. Western blot was utilized to evaluate the efficiency of stathmin 1 overexpression. SPSS 22.0 software was used to show the data as the mean and standard deviation (SD) values (IBM Corporation, USA). The Kolmogorov-Smirnov test was utilized to estimate the conformity of the data to a Gaussian distribution. Student's t-test or Welch's t-test was conducted to assess the significance of differences between two groups, and one-way analysis of variance or the Mann-Whitney U test was conducted to determine the significance of multigroup differences. Two-tailed p values of less than 0.05 were considered to indicate significant differences.

| The effect of propofol and propofol combined with paclitaxel on the viability of cervical cancer cells
Various clinically relevant concentrations of propofol displayed no effect on the viability of HeLa and CaSki cells after treatment for 24 or 48 h ( Figure 1A,B). Propofol reduced HeLa cell viability in a dose-dependent manner ( Figure 1C). In CaSki cells, 100 and 200 μM propofol had no effect on viability, while 400, 600, and 800 μM propofol decreased cell viability ( Figure 1D).
Propofol-induced anesthesia usually occurs at a concentration of 4-6 mg/L, which is equivalent to 22-34 μM. Therefore, we chose 30 μM as the clinically relevant concentration in the follow-up experiments. Cells were treated with 30 μM and the IC30 of propofol (200 μM for HeLa and 400 μM for CaSki cells) combined with various concentrations of paclitaxel for 48 h. The CCK-8 assay results showed that 30 μM propofol had no influence on the cytotoxic effect of paclitaxel, while propofol at its IC30 enhanced the efficacy of paclitaxel in HeLa and CaSki cells ( Figure 1E,F).
Subsequently, the CI values were calculated to evaluate the interaction between paclitaxel and propofol. As shown in Table 1, the IC30 of propofol and 5 or 10 nM paclitaxel had synergistic effect. Hence, 5 nM paclitaxel was chosen to combine with 30 μM and the IC30 of propofol in further experiments. Propofol (30 μM) showed no effect on the efficacy of paclitaxel in HeLa and CaSki cells (Figure 2A,B). In addition, 200 μM propofol (HeLa cells) and 400 μM propofol (CaSki cells) suppressed cell growth and enhanced the cytotoxic effect of paclitaxel ( Figure 2C,D p<0.05).

| The effect of propofol combined with paclitaxel on apoptosis in cervical cancer cells
Annexin-V/PI double staining was utilized to identify apoptotic cells. In sharp contrast to the control treatment, paclitaxel enhanced apoptosis in HeLa and CaSki cells (Figure 3, p < 0.05). However, 30 μM propofol did not influence the promoting effect of paclitaxel on apoptosis. As the concentration increased, propofol accelerated apoptosis in HeLa and CaSki cells and facilitated the apoptotic effect of paclitaxel on these cells (p < 0.05).

| The effect of propofol combined with paclitaxel on the invasion and migration of cervical cancer cells
Transwell assays were conducted to analyze the effect of the two drugs on cell migration and invasion. Propofol amplified the inhibitory effect of paclitaxel on stathmin 1 expression in cancer cells.

| The role of stathmin 1 in the effect of propofol on the efficacy of paclitaxel
To investigate the role of stathmin 1 in the effect of propofol, we established stathmin 1-overexpressing CaSki cells ( Figure 6A) and found that propofol reduced the stathmin 1 protein level ( Figure 6B). High expression of stathmin 1 weakened the inhibitory effect of paclitaxel on cell viability, while propofol treatment partially reversed this effect ( Figure 6C p<0.05). In addition, stathmin 1 impaired the proapoptotic effect of paclitaxel, but the addition of propofol partially restored this effect of paclitaxel ( Figure 6D p<0.05).

| DISCUSSION
Anesthesia is an essential requirement in radical tumor resection surgery. To date, many studies have demonstrated that anesthetics could interfere with cancer development and chemoresistance. 15 Our previous study showed that sevoflurane promoted the malignant behavior of cervical cancer cells but had no effect on cisplatin sensitivity. 16 In addition, isoflurane upregulated the expression of miR-216 to attenuate the malignant progression of colorectal cancer cells. 17 Propofol is a widely used intravenous anesthetic, and increasing evidence has shown that at clinically relevant concentrations (10-100 μM), propofol significantly suppresses the malignant biological behaviors of cancer cells. [18][19][20] Du et al. 21 demonstrated that 30-100 μM propofol reduced the viability of CaSki and SiHa cells in a dose-and time-dependent manner. However, our study demonstrated that 0-100 μM propofol had no effect on cell viability after treatment for 24 and 48 h and that 30 μM propofol did not affect the malignancy of HeLa and CaSki cells after treatment for 48 h. Mitosis is a necessary pathway for cancer cell proliferation. The normal dynamic balance of microtubule polymerization and depolymerization is needed during mitosis to help chromosomes move to the poles via the tension generated by microtubule depolymerization and the thrust generated by microtubule polymerization. 22 Paclitaxel, considered as a microtubule stabilizer, promotes the formation of tubulin dimers and stabilizes existing microtubules, and hinders their degradation to advance microtubule assembly. 23 Owing to the excessive stability of microtubules, the cell cycle is blocked in the late G2 stage, and cell replication is suppressed. 24 β-Tubulin is bound strongly by paclitaxel to stabilize microtubule filaments. 25 A study demonstrated that βtubulin mutations could increase the chemoresistance of cancer cells to paclitaxel. 26 In the present study, paclitaxel treatment increased the level of βtubulin in cervical cancer cells. β-Tubulin expression was not changed after propofol treatment, and the βtubulin protein expression level in the cotreatment group was not different from that in the paclitaxel group, indicating that propofol had no effect on βtubulin expression.
A phosphorylated protein commonly found in the cytoplasm in vertebrates, the stathmin 1 protein impedes the assembly and induces the disassembly of microtubules to take part in the process of mitosis. 27 Various studies have shown that the stathmin 1 protein plays a pivotal role in the evolution of cancer. [28][29][30] For example, the expression of stathmin 1 was negatively related to the degree of tumor differentiation, and a high level of stathmin 1 tended to indicate distant metastasis in pancreatic cancer. 31 Due to the mechanism of paclitaxel in cancer cells, stathmin 1 is a potential drug target of paclitaxel. A study showed that downregulation of stathmin 1 reduced the malignancy of lung cancer cells and decreased the chemoresistance of cancer cells to paclitaxel. 32 In patients with endometrial cancer, stathmin 1 was identified as a biomarker for unfavorable survival, but paclitaxel treatment might improve the outcomes of patients with high levels of stathmin 1 expression. 33 The combination of paclitaxel treatment and silencing of stathmin 1 enhanced the tumoricidal effect compared with that of paclitaxel alone, and paclitaxel suppressed the expression of stathmin 1 in nasopharyngeal carcinoma. 34 In the present research, paclitaxel decreased the expression of stathmin 1, and propofol at high concentrations enhanced the effect of paclitaxel on stathmin 1 expression in cervical cancer cells, indicating that propofol and paclitaxel synergistically suppress the malignancy of cancer cells by downregulating the expression of stathmin 1.
There are some other mechanisms underlying the effect of propofol on the efficacy of paclitaxel. It was reported that propofol might reduce SLUG expression to weaken the proapoptotic effect of paclitaxel in ovarian cancer. 35 Yang et al. 6 found that propofol elevated paclitaxel sensitivity by modulating HOTAIR expression. In addition, a study demonstrated that propofol enhanced the tumoricidal ability of paclitaxel partially by inducing ferroptosis. 36,37 In the present study, propofol might inhibit stathmin 1 expression to enhance the efficacy of paclitaxel.
In conclusion, propofol at high concentrations enhanced the cytotoxicity of paclitaxel, resulting in reduced malignancy of cervical cancer cells, by downregulating stathmin 1 expression, while propofol at clinically relevant concentrations had no effect on the efficacy of paclitaxel in cervical cancer cells; therefore, propofol at clinically relevant concentrations might not affect the response to paclitaxel in patients with recurrent or advanced cervical cancer.