Borneol promotes autophagic degradation of HIF-1α and enhances chemotherapy sensitivity in malignant glioma

Chemicals

Borneol was obtained from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China), and Dulbecco’s Modified Eagle Medium (DMEM) was purchased from ThermoFisher Biochemical Products Co., Ltd. (Gibco, Waltham, MA, United States). Temozolomide and Chloroquine (CQ) was obtained from Selleck Co., Ltd. (Houston, Texas, United States). TdT In Situ Apoptosis Detection Kit - Fluorescein was purchased from R&D Systems, Inc. (Minneapolis, MN, United States). Cell Signaling Technology (Danvers, MA, USA), Affinity Biosciences (Jiangsu, China), ZEN-BIOSCIENCE (Chengdu, China) and MULTISCIENCESL (Hangzhou, China) supplied the antibodies. A CCK8 Kit was purchased from Shanghai Life iLab Biotech Co., Ltd. (Shanghai, China).

Cell proliferation test

C6 cells were obtained from the Laboratory of Traditional Chinese Medicine Preparations, Zhejiang Chinese Medical University. U251 cells were obtained from Wenzhou Medical University. U251 and C6 cells were cultured at 37 °C in a 5% CO2 incubator in DMEM supplemented with 10% FBS, as well as penicillin and streptomycin (100 U/mL). Cells were seeded in 96-well plates and treated for 48 h with 0–800 µM TMZ and/or 0–80 µg/mL borneol. The cell viability of U251 and C6 cells was determined using CCK8 assay. Cell proliferation was analyzed by measuring the absorbance at 450 nm using a multifunctional enzyme marking instrument (Thermo Scientific™ Varioskan™ LUX; Thero Fisher Scientific, Waltham, MA, USA). The cell activity decreased considerably in a concentration-dependent manner (Fig. 1A). For C6 cells, when the TMZ concentration exceeded 250 µM, the decline in survival rate slowed remarkably. Similarly, for U251 cells, the IC50 reached approximately 200 µM (Fig. 1A). Additionally, based on the results of joint drug screening using a gradient of TMZ and borneol (Fig. 1C), the cell viability decreased considerably when the borneol concentration was 40 µg/mL. Therefore, we determined that combined treatment concentrations would involve borneol concentrations of 40 µg/mL, and TMZ concentrations of 200 µM (U251), and 250 µM (C6).

Clonogenic assay

One thousand cells were added into each well and incubated in a CO2 incubator. After 1 week of incubation, when visible colonies formed, the 6-well plates were removed, the culture medium was discarded, and the wells were rinsed once with phosphate-buffered saline (PBS). Methanol was added to each well, and the plate was fixed for 30 min. After discarding the methanol, 0.1% crystal violet (Solarbio, Beijing, China) was added and the cells were stained for 10 min, then washed.

Western blotting (WB)

Cell culture medium was collected, cells were lysed, total protein was extracted, and protein was separated using 8% and 12% SDS-PAGE. The proteins were then transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked with 5% skim milk for 1.5 h at room temperatureat. Blots were incubated with the primary antibodies overnight, washed, incubated with secondary antibodies for 1.5 h, and washed again. Enhanced chemiluminescence (ECL) was used for color development and exposure. Bands detected using WB were scanned in grayscale mode using a multifunction imager (Thermo Fisher Scientific, Waltham, MA, USA). The protein expression of HIF-1α, Beclin-1, and LC3 was then detected. Finally, ImageJ software was used to quantify the WB bands. The antibodies used in this experiment were as follows: HIF-1α (1:1000; Affinity), Beclin-1 (1:1000; Affinity), LC3A/B (1:1000; CST), and GAPDH (1:3000; ZENBIO), Goat Anti-Rabbit IgG(H+L) HRP (1:20000; MULTISCIENCES)

In vivo antitumor activity

SPF-grade male SD rats were obtained from Zhejiang Chinese Medical University Laboratory Animal Research Center. All animal experiments were performed in compliance with guidance of the Zhejiang Chinese Medical University Animal Care and Use Committee (IACUC-20211018-14). After 2 weeks of acclimation, animals were inoculated with C6 cells (1.0 × 10⁶ cells for each rat) using the brain stereotaxic method to establish a glioma model (Zhang et al., 2011). One week after modeling, rats were randomly divided into four groups as follows: control, borneol (16 mg/kg), TMZ (50 mg/kg), and combined treatment group (six rats each). Rats in each group were intragastrically administered the drug daily for nine consecutive days. The general condition of rats as well as the weight of tumors were recorded, and Hematoxylin-Eosin (HE) staining was used for identification.

Transmission electron microscopy (TEM)

Rat brain glioma tissues were first fixed with glutaraldehyde, rinsed in buffer, fixed with 1% osmium acid, washed, and dehydrated in an alcohol gradient, soaked, embedded, polymerized, stained based on sections, and observed via TEM (Hitachi-7650).

Immunohistochemistry (IHC)

Rat brain glioma tissues samples were fixed in paraformaldehyde, dehydrated, and embedded in paraffin. Sections were dewaxed and hydrated, repaired in sodium citrate buffer for 4 h (60 °C oven), inactivated by 3% H₂O₂ for 15 min, washed with distilled water, and incubated with primary antibodies overnight at 4 °C. The sections were then washed, incubated with secondary antibodies, and rinsed with PBS. Diaminobenzidine was used for development, and the slides were rinsed with tap water, stained with hematoxylin, and sealed with a neutral resin. For light microscopy (Leica microscope; Leica, Wetzlar, Germany), the areas and positively stained cells were counted in six random non-overlapping fields of view at 400 × magnification.

TUNEL assay

Rat brain glioma tissues were fixed in paraformaldehyde, dehydrated, and embedded in paraffin. The sections were then dewaxed and hydrated. The samples were covered with 50 µL of Proteinase K Solution and incubated at 37 °C for 30 min. These were then washed twice with distilled water. Next, the slides were immersed in 1X TdT Labeling Buffer for 5 min. The samples was covered with Labeling Reaction Mix and incubated at 37 °C for 1 h in a humidity chamber. To stop the labeling reaction, the samples were immersed in 1X TdT Stop Buffer for 5 min at room temperature. These were then washed twice with PBS for 5 min each at room temperature. Next, the samples were covered with Strep-Fluorescein Solution and incubated for 20 min at room temperature in the dark. After washing with PBS, the slides were sealed with DAPI-containing mounting tablets. The samples were observed and images were captured using a confocal microscopy (Leica confocal microscopy).

Statistical analysis

GraphPad 8 was used for statistical analysis. The number of animals used for the in vivo models in this study was determined based on previous calculations, and the in vitro assays were repeated at least three times. Data are expressed as the mean ± SEM (x ± s), and differences between groups were compared using the t-test. Statistical significance was set at P < 0.05.

Borneol treatment enhances the efficacy of TMZ in glioma

Cell viability was assessed based on a CCK8 assay. C6 and U251 cells were treated with varying doses of TMZ for 48 h. The cell viability assay decreased significantly in a concentration-dependent manner (Fig. 1A). Moreover, U251 cells were more sensitive to TMZ than C6 cells. For C6 cells, when the TMZ concentration was higher than 250 µM, the decline in the cell viability rate slowed significantly, whereas for U251, when the concentration was 200 µM, the viability almost reached the IC50. As shown in Fig. 1B, borneol treatment alone mildly inhibited the growth of C6 cells and U251 cells in the concentration range of 10 µg/mL to 80 µg/mL. In addition, for C6 cells, a significant difference was observed based on borneol concentration up to 40 µg/mL compared to that in the controls (P < 0.05). The results of drug concentration screening, based on TMZ and borneol gradient, showed that the activity of C6 cells decreased remarkably under the combined treatment of 250 µM TMZ and 40 ug/mL borneol (Fig. 1C), compared with other borneol concentrations. For U251 cells treated with 200 µM TMZ, the activity of the cells showed a relatively large decreasing trend when combined with 40 ug/mL borneol, compared with other borneol concentrations.

Subsequently, the effect of borneol combined with TMZ on colony formation was measured in C6 and U251 cells. The results showed that colony formation was reduced. Cell death was increased in the combined treatment group compared to those in the group treated with the same concentration of TMZ alone (Fig. 1D). However, treatment with borneol alone had no significant effect on colony formation. In conclusion, these results suggest that combined treatment with borneol can enhance the cytotoxicity of TMZ in C6 and U251 cells and inhibit cell proliferation.

Effects of borneol and/or TMZ on C6 and U251 protein expression in C6 and U251 cells

Next, we assessed the effectiveness of borneol treatment combined with TMZ, based on protein expression in C6 and U251 cells, compared to that with TMZ treatment alone. We observed the effects of borneol treatment and TMZ on HIF-1α and autophagy levels by WB experiments. The concentration of autophagy inhibitor (CQ) was determined to be 5 µM (C6) and 10 µM (U251) through cytotoxicity tests (Fig. 2C). Beclin-1 and LC3, as markers, are involved in autophagy. Beclin-1 is also a tumor suppressor, and its expression level is negatively correlated with the activity of tumor cells. Our results showed that both borneol and TMZ alone could reduce HIF-1α expression, whereas their combination further reduced HIF-1α protein expression (Figs. 2A and 2B), consistent with our previous results (Qinglin et al., 2021). Moreover, after the use of autophagy inhibitors, the effect of borneol on promoting HIF-1α degradation was reversed (Figs. 2D–2E). From the perspective of autophagy, compared with those in the control group, Beclin-1 levels were increased upon single treatment, and combined treatment also significantly promoted its expression in U251 cells compared to that with TMZ alone (Figs. 3D and 3E). Meanwhile, in C6 cells, combined treatment tended to increase Beclin-1 expression, and the expression level of Beclin-1 increased as the concentration of borneol increased. However, no significant difference (P = 0.0699) was found between the combined treatment and TMZ treatment alone (Figs. 3A and 3B). Further, the results of our study indicated that the level of LC3 II/I was significantly increased by combined treatment when compared with that in the control group, and combined treatment tended to further increase the level of LC3 II/I compared to that with TMZ treatment alone (Figs. 3C and 3F). In general, our results proved that borneol and TMZ alone could promote autophagy in C6 and U251 cells, as well as HIF-1α protein degradation. The combination of the two further increased the level of autophagy and promoted the degradation of HIF-1α.

Enhanced in vivo anticancer efficiency of TMZ when combined with borneol

Subsequently, an in-situ transplantation model of C6 rat glioma was generated using brain stereolocalization to evaluate the efficacy of borneol in combination with TMZ for the treatment of glioma. Our results suggest that both borneol and TMZ alone could reduce the tumor weight when compared to that in the control group, and that combination treatment inhibited tumors more effectively than TMZ alone, consistent with the results of our in vitro experiments (Fig. 3A). Based on the analysis of body weight of SD rats at the beginning of modeling and during administration (from the 7th day onward), the glioma brain model was successfully created approximately one week after modeling, but there was no significant difference in the influence of different treatments on the body weight of rats (Fig. 4B). Moreover, HE staining revealed that the tumor density of the borneol/TMZ combined treatment was significantly lower compared to that with in separate treatments, indicating that the combined treatment could further inhibit the cell proliferation (Fig. 4D). In addition, TEM revealed that both borneol and TMZ could promote autophagy and that combined borneol /TMZ treatment further stimulated autophagy (Fig. 4C). In conclusion, borneol induces autophagy in glioma cells and enhances the tumor-suppressive effects of TMZ.

IHC evaluation of the effects of borneol and/or TMZ on protein expression in tumor tissue

Figure 5 shows, the immunohistochemical analysis of the tumor tissues. Compared with that in the untreated-tumor bearing group, the level of HIF-1α expression in animal glioma tissues treated with borneol and/or TMZ was significantly reduced (Fig. 5A), and the effect of combined therapy was even greater. In addition, the combined use of borneol and TMZ significantly increased the expression levels of Beclin-1 and LC3A/B in glioma tissues compared to those in the untreated controls (Figs. 5B and 5C). Consistent with this, TEM inspection (Fig. 5B) revealed that compared with that in the untreated control group, a large number of autophagosomes was observed in the borneol, TMZ, and combined treatment groups, and compared to that with TMZ alone, the combined treatment further promoted autophagy levels. In accordance with previous studies, borneol inhibited HIF-1α expression by regulating the mTORC1/eIF4E pathway (Wang et al., 2020b), and we found that borneol might make glioma cells sensitive to TMZ by promoting HIF-1α autophagic degradation.

Effects of borneol and TMZ on apoptosis of tumor cells

We evaluated the effect of borneol on apoptosis by conducting a TUNEL (Fig. 6). Compared to the control group, treatment with borneol alone showed a slightly increase in the number of apoptotic cells. Both TMZ treatment alone and the combined treatment significantly increased apoptosis level. Furthermore, the combined treatment showed a further increase in apoptosis compared to TMZ treatment alone. These experimental results are consistent with those of previous studies (Wang et al., 2020b). Borneol alone can down-regulate HIF-1α and promote apoptosis(Wang et al., 2020b), which further validates our study’s findings that borneol induces apoptosis in glioma cells by promoting autophagy to degrade HIF-1α.