Introduction

Lung cancer is a malignancy with high morbidity and mortality rates [1, 2]. Non-small cell lung cancer (NSCLC) is the most common form, accounting for about 85% of all occurrences [3]. Progress has been made in NSCLC treatment using surgery, chemotherapy, radiation, and molecularly targeted therapy, yet 5-year overall survival rate remains poor [4, 5]. Platinum-based chemotherapy is recommended as a first-line therapeutic option. Cisplatin (CDDP) is widely used clinically [6, 7], and is a key drug for treating multiple human cancers, including NSCLC. The drug acts by damaging DNA and inducing apoptosis [8, 9]. However, acquired resistance inexorably develops after a period of treatment and remains a major limitation to clinical application [10,11,12]. Synergistic combinations of drugs enhance therapeutic efficacy and are often used to overcome drug resistance. Thus, new and safe combinations to improve NSCLC cell sensitivity to CDDP are critical to successful treatment.

Combinations of agents derived from natural products with antitumor drugs have gained attention mainly owing to their ability to prevent cancer development and progression by inhibiting proliferation, differentiation, metastasis, and angiogenesis, or by inducing apoptosis and regulating autophagy and the cell cycle. These combinations aim to improve therapeutic efficacy, decrease the risk of adverse side effects and toxicity, and overcome the acquired drug resistance [13,14,15,16].

Borneol, a monoterpenoid isolated from several medicinal plants, including Blumea blasamifera (L.) DC. and Cinnamomum camphora (L.) Presl, is a characteristic traditional Chinese medicine (TCM) resuscitation agent. The compound can awaken and refresh and has been used for cardiovascular and cerebrovascular diseases for thousands of years. The Chinese Pharmacopoeia separates borneol into three categories: natural borneol (dextrorotatory borneol, d-borneol), levorotatory borneol (l-borneol), and synthetic borneol (SB), to reflect differences among sources [17]. Borneol is widely regarded as a messenger drug capable of delivering main effective drugs in the prescription to the target site to improve therapeutic efficacy. Borneol can enhance drug penetration across different physiologic barriers, such as the blood–brain barrier (BBB) [18], cornea [19], skin [20], and mucous membrane [21]. This property encourages increased blood concentrations and bioavailability of other drugs. d-Borneol seems safer than SB [22]. Further, borneol may prevent drug efflux by suppressing P-glycoprotein (P-gp) function and acting as a chemotherapeutic sensitizer to promote therapeutic efficacy [23]. For example, borneol sensitizes cancer cells to chemotherapeutic drugs, such as cisplatin [24], curcumin [25], paclitaxel [26], and doxorubicin [27], and enhances the effects of therapies for glioma, melanoma, hepatocellular carcinoma, and esophageal squamous cell carcinoma. However, the impact of d-borneol on NSCLC cells and its ability to stimulate chemosensitivity to CDDP in resistant cells is not well studied, and underlying processes remain unclear. Therefore, we initially selected an NSCLC cell line sensitive to d-borneol and established optimum concentrations and exposure durations. We used the corresponding cisplatin-resistant cell line, H460/CDDP, to investigate synergy between d-borneol and CDDP on proliferation, cell cycle progression, and apoptosis.

RNA sequencing (RNA-seq) is an approach for whole-transcriptome sequencing to analyze key factors of d-borneol-induced sensitization. We focused on cyclinA2/D3-CDK2/6 signaling pathway-related cell cycle arrest, the Bax/Bcl-2/caspase3 pathway associated with apoptosis, and P-gp function to attempt to explain mechanisms of synergy between d-borneol and CDDP. Lastly, we explored antitumor and major protective effects of d-borneol with CDDP in vivo.

Materials and methods

Cell lines and reagents

Human lung cancer A549 (CAT: FH0045) (Additional file 1) and H460 (CAT: FH0050) (Additional file 2) cells were kindly provided by Dr. Cao (Chengdu University of Traditional Chinese Medicine, Chengdu, China), and H460/CDDP cells were purchased from FuHeng Biology (CAT: FH1207, FuHeng Cell Center, Shanghai, China). At 37 °C in a humidified environment with 5% CO2, A549, NCI-H460, and H460/CDDP cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Gibco, Australia) with 10% fetal bovine serum (FBS; Gibco, Australia) and 1% penicillin/streptomycin solution (Gibco, Australia) added. CDDP was obtained from Sigma-Aldrich (St. Quentin Fallavier, France). d-Borneol (#T5734) with purity (99.6%) was purchased from Shanghai Topscience Co., Ltd. DMSO (D8371) was purchased from Solarbio Science & Technology Co., Ltd. MTT reagent was purchased from BioFroxx. RT EasyTM I and Real Time PCR Easy SYBR Green One-Step Kit were purchased from Foregene Co., Ltd. Anti-Bax (T40051), Bcl-2 (T40056), and caspase-3 (M005851) were purchased from Abmart (Shanghai, China). Anti-cyclinA2 (ab181591), CDK2 (ab32147), and p27 (ab32034) were purchased from Abcam (Paris, France). Anti-p21 (AF6290), cyclinD3 (AF6251), CDK6 (DF6448), P-gp (AF5185) and tubulin (AF7011) were obtained from Affinity Biosciences.

Determination of borneol type and cell line

The cytotoxicity of borneol on H460 and A549 cells was determined by MTT. Cells were seeded at a density of 5 × 103 cells per well in 96-well plates and incubated for 24 h or 48 h. Then, cells were treated with different doses of d-borneol, l-borneol, and SB (0, 1, 2, 4, 8, 16, and 32 μg/ml) prepared in DMSO and diluted in RPMI-1640 with 1% FBS. After 24 h or 48 h, 20 μl MTT reagent was added to each well. Then, the reagent was removed after incubation for 4 h at 37 °C, and 100 μl DMSO was added. Absorbance at 570 nm was measured by Bio-Tek microplate reader after incubation for 10 min (BioTek, Winooski, VT, USA). Cell viability = (treated viable cells)/(control viable cells) × 100%.

Cell viability assays of combined treatment

To evaluate the concentration of CDDP, H460 and H460/CDDP cells (5 × 103 per well) were seeded in a 96-well plate and treated with CDDP (0.625–160 μg/ml) for 24 h. Then H460/CDDP cells were exposed to CDDP (0.625–160 μg/ml) for 24 h and 48 h to determine the suitable time. Next H460/CDDP cells was exposed to three borneol forms (0.5–16 μg/ml) for 24 h to determine the concentration and type of borneol. Finally for combined treatment, H460/CDDP cells were pretreated with d-borneol (0.5, 1, 2, and 4 μg/ml) for 12 h and co-incubated with CDDP (10 μg/ml) for 24 h. MTT assay, as described in “Determination of borneol type and cell line” section, was used to determine cell viability. The morphology of the cells was observed and photographed by inversion fluorescence microscopy (Leica DMI3000B, Germany).

Evaluation of P-gp function assay

H460/CDDP cells were incubated with CDDP (10 μg/ml) plus d-borneol (0.5, 1, 2, and 4 μg/ml) for 24 h. The cells were resuspended in the medium, stained with rhodamine 123 (Rho 123) solution (Beyotime Biotechnology, Shanghai, China), and incubated for 30 min at 37 °C in 5% CO2. The cells were centrifuged for 5 min at 2000 rpm, then resuspended and incubated for 120 min after being washed twice with medium. Then the cells were centrifuged again and washed twice with PBS. A fluorescence spectrophotometer was used to measure the fluorescence of Rho 123 with emission wavelength of 529 nm and excitation wavelength of 507 nm (BioTek, Winooski, VT, USA).

Scratch-wound healing recovery assays

A monolayer of H460/CDDP cells was scratched with the tip of a 100 μl pipette for the wound healing experiment, and the floating cells were rinsed off with PBS. Under a microscope, the gap widths were measured. After that, the cells were treated with different doses of CDDP (10 μg/ml) and d-borneol (0.5, 1, 2, and 4 μg/ml), and photographs of the wounds were taken in four random areas by microscope (Leica, Germany) at 0 h and 24 h. ImageJ was used to measure the scratch areas.

Colony-formation assay

H460/CDDP cells were planted at a density of 2 × 105 cells per well in six-well plates. The cells were next treated with CDDP (10 μg/ml) for 24 h, or with CDDP (10 μg/ml) with d-borneol (0.5, 1, 2, and 4 μg/ml) for 24 h. Cells were then digested with trypsin, and 500 cells were reseeded onto six-well plates and cultured for a further 8–10 days. Finally, crystal violet staining solution was used to stain the colonies (Beyotime Biotechnology, Shanghai, China). The number of colonies was quantified by ImageJ. The proportion of control was used to normalize the results.

Flow cytometry cell cycle analysis

H460/CDDP cells were logarithmically cultivated and seeded into 6-cm dishes at a density of 2 × 104 cells/ml. After treatment with the combination of d-borneol (0.5, 1, 2, and 4 μg/ml) and CDDP (10 μg/ml) for 24 h, H460/CDDP cells were collected, washed with PBS, and fixed with ethanol for 24 h. Then the cells were stained with propidium iodide (PI) for 30 min and protected from light. Flow cytometry was utilized to detect changes in cell cycle distribution (BD Biosciences, CA, USA) and analyzed by FlowJo V10 software [30].

Borneol, a traditional Chinese medicine, resuscitates and refreshes, and is usually used to treat cerebrovascular diseases. Natural borneol can boost the antitumor properties of selenocysteine [31] and curcumin [32] in hepatocellular carcinoma. However, whether borneol enhances sensitivity to chemotherapeutic drugs in NSCLC, and its underlying mechanism, is still unclear. Moreover, few studies compare the effects of three forms of borneol (d-borneol, l-borneol, and SB) in parallel on NSCLC cells. Therefore, this study is the first to investigate these compounds in lung cancer cells, identify the most sensitive cell lines and borneol forms, focus on the synergistic effects of borneol with CDDP, and systematically investigate chemosensitization mechanisms using RNA-seq analysis.

Multidrug resistance (MDR) usually manifests owing to the overexpression of P-gp, the main factor limiting response to chemotherapy in many cancers [33]. Inhibition of drug efflux mediated by P-gp might resensitize resistant cancer cells to chemotherapy [34]. Natural products are known to enhance the cytotoxicity to drug-resistant cells by physically inhibiting P-gp activity [35]. This action is consistent with our results, which indicate that d-borneol sensitized H460/CDDP cells to CDDP by inhibiting P-gp expression and function.

RNA-seq is a high-throughput sequencing technology with the advantages of rapid analysis and high resolution. RNA-seq analysis showed that the downregulated genes were mainly enriched in cell cycle. Arrest at any cycle phase will cause cell growth stagnation or death. The cell cycle is a continuous process and sensitive to disruption [36]. Further, altering the regulation of cell cycle progression is a crucial strategy for eliminating cancer cells [37, 38]. CDK kinase complexes are formed and activated by sequential activation and inactivation of CDK and cyclins at specific stages of the cell cycle. This sequential activation and inactivation is necessary for progress through the cell cycle [39]. Many natural products can block the cell cycle at different phases in various cancer cell lines. This block is caused by downregulating the expression of cyclin A, D, and E subunits and reducing the activity of cyclin/CDK [40,41,42]. Cyclin D, in particular, is associated with CDK4/6 and is the key regulator of G1-to-S transition [43]. S phase is the DNA replication stage, which is the key part of the entire cell cycle. Complexes formed by cyclin A/cyclin E and their respective kinase (CDK2) promote the progression through S phase, and downregulation of these cyclins and CDKs results in the accumulation of cells in S phase [44, 45]. The cyclin A/CDK2 complex affects both the beginning of DNA synthesis and the S-phase progression [46]. Flow cytometry showed that cotreatment with d-borneol and CDDP increased cell number in S phase but decreased G0/G1 phase, suggesting that d-borneol arrested the cell cycle at S phase. Further, western blotting showed that protein expression of CDK2, CDK6, cyclin A, and cyclin D decreased, consistent with the results of flow cytometry. Thus, d-borneol enhanced sensitivity to CDDP in H460/CDDP cells by modulating CDKs and cyclins associated with S-phase arrest.

CKI is an upstream regulator of CDKs and cyclins and is vital for cell cycle progression [47]. p21 and p27 are universal CKIs that physically bind to and inhibit functions of cyclin–CDK2, cyclin–CDK1, and cyclin–CDK4/6 complexes. This inhibition ultimately leads to the interruption of the cell cycle in G1 and S phases [48, 49]. Downregulation of p21 is linked to tumor differentiation, invasion, proliferation, and metastasis in several studies [50, 51]. Moreover, targeting p21 is a potential strategy for the treatment of tumors. Abnormal expression of p21 and p27 proteins is critical for the appearance of drug-resistant phenotypes following cancer therapy [52]. d-Borneol and CDDP together induced upregulated expression of mRNA and protein levels of p21 and p27 to levels that exceeded those seen for CDDP used alone. Similarly, Liu et al. [53] reported that the upregulation of p21 in lung cancer cells arrested the cell cycle, inhibited cell proliferation, and increased sensitivity to cisplatin. Taken together, we have shown that d-borneol combined with CDDP induced cell cycle arrest at S phase by upregulating p21 and p27 and suppressing cyclin A2/CDK2 and cyclin D3/CDK6 to enhance CDDP sensitivity in H460/CDDP cells through RNA-seq.

Cell apoptosis is critical for cancer prevention and therapy [54]. Apoptosis is mediated by two primary pathways: the mitochondria-driven apoptotic intrinsic pathway and the death receptor-induced extrinsic pathway [55]. p21 binds to Bcl-2 family proteins to release Bax proteins through the formation of a p21/Bcl-w complex, thus promoting cell apoptosis and inhibiting cell invasion [56]. The Bcl-2 family, which includes pro-apoptotic members (Bax, Bad, and Bak) and anti-apoptotic members (Bcl-2, Bcl-xL, and Bcl-B), plays a major role in the initiation of the intrinsic apoptotic pathway [57]. Caspases are a cysteine protease family and crucial effector molecules in caspase-dependent apoptosis. Activation of caspase-3 may initiate cell apoptosis [58]. Our results indicated that d-borneol and CDDP cotreatment promoted cell apoptosis and thus enhanced the sensitivity of CDDP to H460/CDDP cells via p21-mediated Bax/Bcl-2/caspase-3-activated intrinsic cell apoptosis.

To confirm the antitumor activity of d-borneol combined with CDDP in vivo, H460/CDDP cells were subcutaneously transplanted into nude mice. Combined treatment of d-borneol and CDDP after 14 days significantly inhibited tumor volume and weight while causing no obvious decrease in body weight, compared with the CDDP group. Interestingly, serum levels of ALT, AST, BUN, and CRE in the CDDP group were significantly increased, accompanied by obvious liver and kidney histopathological damage. Many inflammatory and necrotic cells were seen in liver and kidney tissue, edematous renal tubular epithelial cells were observed, and damaged casts appeared in kidney. However, no damage was observed in tissues from mice treated with CDDP and d-borneol. CDDP damaged in liver and kidney is thus ameliorated by combined treatment. Thus, animals that received both agents showed reduced tumor growth, and also less liver and kidney toxicity. This finding demonstrates the advantage of the combination treatment for increasing efficacy and reducing toxicity.

Conclusions

We showed for the first time that H460 cells exhibit higher sensitivity to d-borneol than A549 cells. Further, combined application of d-borneol and CDDP enhanced cell sensitivity through activating intrinsic apoptosis via p21-mediated Bax/Bcl-2/caspase-3 signaling and arresting the cell cycle at S phase via p27/p21-mediated cyclinA2/D3-CDK2/6. These findings support the reliability of RNA-seq results and our initial hypothesis. Our study has also found that d-borneol reduced the expression of P-gp and inhibited drug efflux, thereby increasing sensitivity to CDDP. In vivo, d-borneol combined with CDDP inhibited NSCLC tumor growth significantly and ameliorated CDDP toxicity (Fig. 8), which reflects the advantages of increasing efficacy and reducing toxicity by combining natural products and chemotherapeutic drugs. Biological mechanisms of tumor occurrence and development are complicated, and much is still unknown. Therefore, we will focus on the key biological mechanisms in vivo to provide experimental evidence that d-borneol is potential chemosensitizer for the treatment of lung cancer.

Fig. 8
figure 8

Schematic diagram of antitumor effect in vivo and in vitro and the proposed signaling pathway induced by combination treatment with d-borneol and CDDP. d-Borneol enhanced the sensitivity of CDDP by increasing CDDP cellular accumulation via inhibiting P-gp function. On the one hand, d-borneol enhanced p21 and p27 and suppressed two complexes, i.e., CDK2–cyclin A and CDK6–cyclin D, which blocked the cell cycle in S phase. On the other hand, d-borneol enhanced the sensitivity of CDDP to H460/CDDP cells by promoting the mitochondrially mediated intrinsic apoptosis via activating pro-apoptotic Bax, downregulating anti-apoptotic Bcl-2, and finally promoting activation of caspase-3, which leads to apoptosis. “←” indicates activation, and “⟝” indicates inhibition

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