Plumbagin Enhances the Anticancer Effects of PF Chemotherapy via Downregulation of the PI3K/AKT/mTOR/p70S6K Pathway in Human Tongue Squamous Cell Carcinoma

Cisplatin plus 5-fluorouracil (PF) is used as the standard neoadjuvant chemotherapy (also called preoperative chemotherapy) in the treatment of tongue squamous cell carcinoma (TSCC). Although PF chemotherapy reduces the distant metastasis of TSCC, the five-year survival rate has not significantly improved. In recent years, components considered in traditional Chinese medicine have been researched as adjuvant drugs for radiotherapy and chemotherapy. Plumbagin (PB) is a quinone component isolated from Plumbago zeylanica L. Notably, PB demonstrates numerous anticancer properties. In order to examine the chemosensitization effect of PB on PF and its associated mechanisms, in vitro experiments using TSCC Cal27 and cisplatin (CDDP)-resistant Cal27/CDDP cells were carried out in the present study, and the results were subsequently verified using nude mice xenografts. Results of the present study demonstrated that PB enhanced the anticancer effects of PF on the proliferation, migration, and invasion of Cal27 and Cal27/CDDP cells. Cell cycle assays demonstrated that both Cal27 and Cal27/CDDP cells were arrested in the S phase following the combined treatment of PF and PB. Moreover, the PF and PB combination group induced higher levels of apoptosis in Cal27 and Cal27/CDDP cells compared with the group treated with PF alone. In addition, the results of the present study demonstrated that combined PB and PF inhibited the PI3K/AKT/mTOR/p70S6K pathway in TSCC cells. Moreover, the weight and volumes of tumors in nude mice were reduced following treatment with a combination of PF and PB. Results of the present study also demonstrated that the expression levels of Ki67 were markedly reduced in the combined treatment group compared with the group treated with PF alone. In summary, the results of the present study demonstrated that PB enhanced the PF sensitivity of TSCC through induction of S-phase arrest and apoptosis via the PI3K/AKT/mTOR/p70S6K pathway.


Introduction
Tongue squamous cell carcinoma (TSCC) is the most common malignancy of the head and neck region, with a high rate of regional lymph node metastasis and high morbidity and mortality [1,2]. Notably, the fve-year survival rate of TSCC is between 48.0 and 70.9%, and the incidence of recurrence following curative treatment of TSCC is 26.5-51.0% [3,4]. Neoadjuvant chemotherapy, as a key method for the treatment of TSCC, plays an important role in controlling tumor size and preserving oral function [5]. In the neoadjuvant chemotherapy regimen for TSCC, cisplatin plus 5fuorouracil (PF) is the standard option. Although neoadjuvant chemotherapy reduces distant metastases in patients with TSCC, fve-year survival rates have not been signifcantly improved [6,7]. Te main factors afecting efcacy are the decreased sensitivity of tumor cells to chemotherapeutic drugs and the emergence of chemoresistance [8]. Terefore, the discovery of novel methods to improve the sensitivity of TSCC to PF chemotherapy is required.
In recent years, an increasing number of researchers have focused on treatment options derived from natural plants [9,10]. Plumbagin (PB) is a quinone component isolated from the rhizome of Plumbago zeylanica L., which exerts antioxidant, anti-infammatory, antibacterial, anticancer, and other biological activities [11]. In vitro and in vivo experiments demonstrated that PB exhibits antitumor properties in various cancers, such as breast cancer, pancreatic cancer, and lung cancer, with no notable toxicity observed in healthy cells [12]. Notably, PB demonstrates antiproliferative [13], antimetastatic [14], proapoptotic [15], and proautophagic cell death [16] efects in both tumor cells and animal models. Results of our previous studies demonstrated that PB might induce reactive oxygen species (ROS) and apoptosis in TSCC cells by targeting multiple signaling proteins including MAPKs and AKT/mTOR [17,18]. However, the sensitization efect of PB on chemotherapeutic drugs and the associated mechanism are yet to be fully elucidated.
Te present study aimed to explore the sensitization role and mechanisms of PB on PF chemotherapy, using the Cal27 and cisplatin-resistant Cal27/CDDP TSCC cell lines. We found that PB enhanced the anticancer efects of PF on the proliferation, migration, and invasion of Cal27 and Cal27/ CDDP cells. In vivo study revealed that PB and PF in combination signifcantly inhibited TSCC xenograft tumor growth.

Cell Line and Culture
Conditions. TSCC cell lines (Cal27) and TSCC chemoresistant cell lines (Cal27/CDDP) were granted by Sun Yat-sen Memorial Hospital (Guangzhou, China). Te chemoresistant cell line Cal27CDDP was created by exposing Cal27 cells to cisplatin at the indicated time [19]. Both of the aforementioned TSCC cells were cultured in DMEM containing 10% (v/v) FBS at 37°C in a humidifed incubator with 5% CO 2 .

Cell Viability and Combination Index (CI) Analysis.
Te CCK-8 test was carried out to examine the efects of diferent concentrations of PB, and/or 5-Fu, and/or CDDP on cell viability. Cal27 and Cal27/CDDP cells were cultured in 96-well plates for 24 h with a density of 5 × 10 3 . Ten, the cells were treated with PB (1.25-20 μM) and/or 5-Fu (160-2560 μg/ml) and/or CDDP (2.5-40 μg/ml) for 24 h to determine the individual and combined efects of PB and PF. Subsequently, 10 μl CCK-8 solution was added to each well, and the plates were incubated for 2 h. Te optical density (OD) was measured at 450 nm in a Spark TM microplate reader (Tecan, Inc., Switzerland). Cell viability was calculated as a percentage of the control cells. Cell viability (%) � (OD treatment-OD blank)/(OD control-OD blank) × 100%. Te median inhibitory concentration (IC 50 ) was calculated with growth inhibition curves ftted to the data using GraphPad Prism 8.0.
Te combined efects of PB and PF on TSCC cells were evaluated using the CI according to the median dose-efect analysis reported by Chou and Talalay [20]. Fractional inhibition (FA) � 1-fraction of surviving cells. Te corresponding CI values were analyzed using CompuSyn Software. CI < 1 represents synergistic efect; �1 represents additive cytotoxicity; >1 represents antagonistic efect.

Edu Assay.
Cal27 and Cal27/CDDP cells were incubated in 96-well plates with a density of 5 × 10 3 for 24 h. Ten, the cells were treated with 2.5 μM PB or PF (320 μg/ml 5-Fu + 2.5 μg/ml CDDP) alone or PB + PF in combination for another 24 h. Te previous medium was removed, and cells were washed with PBS three times. A total of 100 μl Edu solution (50 μM) diluted with culture medium at the ratio of 1000 : 1 was injected into each well and cocultured for 2 h. Subsequently, 4% paraformaldehyde was used to fx the cells, and Hoechst 33342 was added to stain the DNA. Cells were observed by using an inverted fuorescence microscope.

Colony Formation Assay.
Cal27 and Cal27/CDDP cells were cultured in six-well plates with 800 cells in each well. 2.5 μM PB or PF (320 μg/ml 5-Fu + 2.5 μg/ml CDDP) alone or PB + PF was employed to treat both cell types for 24 h. Following 2-week incubation, cells were washed with PBS three times and fxed in 4% paraformaldehyde solution for 20 min. Cells were subsequently stained with 2 ml Giemsa solution for 30 min. Colonies with more than 50 cells were scored. Colony formation rate � (colony number/seeded cell number) × 100%.
2.6. Transwell Invasion Assay. Te transwell invasion assay was carried out using 24-well plates inserted with 8 μm chamber coated with Matrigel. In the upper chamber, cells were cultured in the FBS-free medium with a density of 3 × 10 4 . A medium containing 20% FBS was injected into the lower chamber. Cells were subsequently treated with 2.5 μM PB or PF (320 μg/ml 5-Fu + 2.5 μg/ml CDDP) alone or PB + PF for 24 h. Cells were fxed under the membrane and stained with 0.1% crystal violet for 10 min. Cell numbers . Mice were housed in a standard specifc-pathogen-free environment. 0.15 ml cell suspension containing 1 × 10 6 Cal27/CDDP cells were injected into the right fank of each mouse. Xenograft tumor growth was monitored daily. When the tumor reached ∼80 mm 3 , 24 mice were divided (n � 6 in each group) into four groups: (i) the control group, mice were injected with 0.9% saline; (ii) the PB group, mice were injected with 3 mg/kg PB every other day; (iii) the PF group, mice were injected with 4 mg/ kg 5-Fu and CDDP every 3 days; and (iv) the PB + PF group, both PB and PF were administrated in combination, in concentrations as previously described. Te tumor volumes and body weights of the mice were measured every day. Tumor volume � a × b 2 /2 (a, the largest diameter; b, the shortest diameter of the tumor). After 21 days, all mice were sacrifced; tumors were then isolated and weighed. 10% formalin was used to fx the heart, liver, spleen, lung, and kidney. After being dehydrated in ethanol and embedded in parafn, a series of parafn sections (5 μm) of the abovementioned organs were stained with hematoxylin-eosin (H and E). All the stained samples were analyzed using a light microscope with 400x magnifcation.
2.12. Immunohistochemistry. All xenograft tumors were excised and fxed in 4% paraformaldehyde for further embedding in parafn. Subsequently, the parafn samples were cut into 4 μm slides. Te slides were blocked and incubated in Ki67 antibody solution (1 : 2000 dilution; 27309-1-AP). Te expression level of Ki67 was examined by counting the number of positive cells from fve randomly selected felds with 400x magnifcation (Olympus BX 53).

Statistical Analysis.
All experiments were performed in triplicate, and the data were presented as the mean-± standard deviation. Multiple comparisons were evaluated using a one-way ANOVA and Tukey's post hoc test. When GraphPad 8.0 software was used for statistical analysis, P < 0.05 was considered statistically signifcant. Tus, concentrations of 2.5 μM PB, 320 μg/ml 5-Fu, and 2.5 μg/ml CDDP were selected for further experiments. Both Cal27 and Cal27/CDDP cells were divided into four groups, namely, (i) the control group, cells treated with medium only; (ii) PB group, cells treated with 2.5 μM PB; (iii) the PF group, cells treated with 320 μg/ml 5-Fu and 2.5 μg/ml CDDP; and (iv) the PF + PB group, cells treated with both PB and PF, concentrations as previously described. Compared with PF alone, treatment with PB + PF signifcantly enhanced the inhibition of Cal27 and Cal27/CDDP cells (Figure 1(d)). According to the median dose-efect analysis by Chou and Talalay, the CI values of the combination of PB and PF were 0.73550 and 0.82662 in Cal27 and Cal27/CDDP cells, respectively (Supplementary Table SI). Tese results demonstrated that the combination of PB and PF exerted synergistic cytotoxic efects in Cal27 and Cal27/CDDP cells. Subsequently, an Edu assay was used to examine the proliferation of TSCC cells. Compared with PF alone, PB + PF treatment markedly decreased the green fuorescence intensity by 87.60% in Cal27 cells and 91.79% in Cal27/CDDP cells (Figures 2(a)-2(c)). A colony formation assay was carried out to examine the colony formation of TSCC cells. Compared with PF alone, PB + PF treatment notably decreased the colonies by 75.06% in Cal27 cells and by 86.42% in Cal27/ CDDP cells (Figures 2(d)-2(f)). Te transwell assay revealed that PB + PF signifcantly reduced the invasion ability of Cal27 and Cal27/CDDP cells (Figures 3(a) and 3(b)). Te wound healing assay demonstrated that PB + PF signifcantly decreased the migration ability of Cal 27 and Cal27/CDDP cells, compared with PF alone (Figures 3(c) and 3(d)). Tese results indicated that PB synergistically enhanced the cytotoxicity of PF in TSCC cells, and the combination of PB and PF inhibited the invasion and migration of TSCC cells.

PB and PF in Combination Promotes S Arrest in TSCC
Cells. Flow cytometry was carried out to examine the cell cycle distribution following the drug treatment. Cal27 and Cal27/CDDP cells were treated with 2.5 μM PB, PF (320 μg/ml 5-Fu and 2.5 μg/ml CDDP), and PB + PF for 24 h. In Cal27 cells, compared with PF alone, PB + PF signifcantly increased the S phase by 1.31-fold (Figures 4(a) and 4(b)). In Cal27/CDDP cells, compared with the PF single group, the PB and PF combination group signifcantly increased the S phase by 3.67-fold (Figure 4(c)). Tis indicated that combined PB and PF could signifcantly induce S phase arrest in TSCC cells.

PB Enhances the Proapoptosis Efect of PF in TSCC Cells.
Subsequently, the present study aimed to determine whether PB afected cellular apoptosis and whether apoptosis is a mechanism of synergism between PB and PF. Cal27 and Cal27/CDDP cells were treated with 2.5 μM PB, PF (320 μg/ml 5-Fu and 2.5 μg/ml CDDP), and PB + PF for 24 h. Annexin V-FITC/PI fow cytometry was used to quantify apoptosis. As shown in Figure 5(a), PB and PF in combination signifcantly induced apoptosis. Compared with PF alone, PB + PF increased cellular apoptosis (both early and late apoptosis) by 2.88-fold in Cal27 cells ( Figure 5(b)) and 2.93-fold in Cal27/CDDP cells ( Figure 5(c)). Te enhanced proapoptosis efect of PF by PB in Cal27/CDDP cells was more potent than that in Cal27 cells. Subsequently, western blot was carried out to explore the potential mechanisms underlying the proapoptotic efects of PB and PF in combination. Te expression levels of apoptosis-related proteins, Bax, Bad, Bcl-2, and Bcl-xL, were examined in Cal27 and Cal27/CDDP cells. As shown in Figures 5(d)-5(g), the combined treatment of PB and PF signifcantly decreased the expression of the antiapoptotic protein Bcl-2 and Bcl-xL while increasing the expression of the proapoptotic proteins, Bax and Bad, in both cell lines, compared with PB or PF treatment alone. Collectively, these results indicated that PB enhanced the apoptosis-inducing efect of PF in TSCC cells.

Combined PB and PF Inhibit the PI3K/AKT/mTOR/ p70S6K Pathway in TSCC Cells.
To further explore the molecular mechanisms underlying PB-enhanced PF chemotherapy in the inhibition of TSCC cells, western blot analysis was carried out to detect the expression of proteins involved in the PI3K/AKT/mTOR/p70S6K signaling pathway, which plays a key role in cell cycle and apoptosis. Compared with the control group, the protein expression levels of AKT, mTOR, and p70S6K in Cal27 and Cal27/ CDDP cells in the PB group were not signifcantly altered. However, the protein expression levels of PI3K, p-AKT, p-mTOR, and p-p70S6K were all decreased. Compared with the PF group, the expression levels of PI3K, p-AKT, p-mTOR, and p-p70S6K in the cells of the PB + PF group were also decreased (Figures 6(a)-6(e)). Tese results indicated that a combination of PB and PF chemotherapy might inhibit the PI3K/AKT/mTOR/p70S6K signaling pathway in TSCC cells.

Combined PB and PF Inhibits TSCC Xenograft Tumor
Growth In Vivo. Based on the synergistic inhibition of Cal27 and Cal27/CDDP cells following treatment PB + PF, the present study aimed to determine whether similar therapeutic efects occurred in a subcutaneous cisplatin-resistant xenograft nude mice model. Cal27/CDDP cells were injected into the right fank of male nude mice. Results of the present study demonstrated that after 21 days of treatment, tumor growth was inhibited in PB or PF or PB + PF groups. Notably, PB + PF exerted the greatest inhibition of tumor volume and weight, compared with either PB or PF treatment alone (Figures 7(a)-7(d)). In addition, the weights of nude mice were signifcantly decreased following the treatment with PF, compared with either PB or PF treatment alone. On the other hand, PB + PF treatment reversed this phenomenon (Figure 7(e)). Tese results indicated that the combined treatment of PB + PF was more efective in tumor control and exerted a lower level of toxicity. In addition, H 4 Journal of Oncology and E staining verifed that PB + PF did not cause toxicity in the major organs, including the heart, liver, spleen, lung, and kidney (Figure 7(g)). Results of the present study also demonstrated that PB + PF signifcantly inhibited Ki67 expression, compared with PF alone (Figure 7(f )). Collectively, these results demonstrated that PB combined with PF might exhibit potential as a strategy to reduce PF resistance in TSCC while also reducing any systematic side efects.

Discussion
At present, neoadjuvant chemotherapy (NACT), systemic chemotherapy prior to surgery, or radiotherapy is used to meet the conditions required for subsequent surgery and radiotherapy to reduce tumor volumes and inhibit metastatic cells [21]. NACT is mainly used in patients with high pathological stage cancer and large tumor volumes. Tus, patients who were previously inoperable may be ofered surgery in order to inhibit potential metastases. Notably, NACT is widely used in a variety of malignant tumors including TSCC. Te currently used NACT regimens include the DBP regimen (pingyangmycin + cisplatin + 5-Fu), the PDF regimen (paclitaxel + cisplatin + 5-Fu), the TPF regimen (docetaxel + cisplatin + 5-Fu), and the PF scheme (cisplatin + 5-Fu), of which PF chemotherapy is regarded as the standard chemotherapy regimen for the treatment of TSCC [22]. However, the toxicity and adverse side efects of chemotherapeutic drugs and the decreased sensitivity of tumor cells to chemotherapeutic drugs greatly afect the efcacy. Terefore, a novel treatment regimen with low toxicity is required to enhance the sensitivity of TSCC to PF.
In recent years, more and more researchers have tended to develop natural herbal extracts due to their fewer adverse side efects and low costs [23]. PB is a kind of quinone extracted from the root of Plumbago zeylanica L., with antiatherosclerosis, anti-infammatory, antibacterial, and antitumor pharmacological activity [11]. In recent years, more and more studies have shown that PB has an excellent killing efect on   Journal of Oncology lung cancer, liver cancer, breast cancer, prostate cancer, and other malignant tumors [24]. Results of the present study demonstrated that PB combined with PF chemotherapy inhibited the proliferation, invasion, and migration of Cal27 and Cal27/CDDP cells. Results of in vivo assays demonstrated that the tumor weight and volume in the combined PB and PF group were reduced compared with cells in the group treated with PF alone. Furthermore, H and E staining verifed that the combined treatment regimen did not cause toxicity in major organs, including the heart, liver, spleen, lung, and kidney. Tese results demonstrated that PB enhanced the therapeutic efect of PF while reducing its toxicity.
Te reported antitumor mechanisms of PB include the elevation of cellular ROS, promotion of apoptosis, cell cycle arrest, and the inhibition of epithelial-mesenchymal transition [25]. Results of the present study demonstrated that PB combined with PF signifcantly inhibited the proliferation of Cal27 and Cal27/CDDP cells by inducing cell cycle arrest in the S phase and promoting cell apoptosis, compared with PF treatment alone. Apoptosis is a type of programmed cell death that can be classifed into intrinsic apoptosis and extrinsic apoptosis. Te mitochondrial pathway is the main regulatory pathway of intrinsic apoptosis [26]. Te Bcl-2 family plays a central role in regulating mitochondrial function and outer membrane permeability. Notably, the Bcl-2 family of proteins can be divided into antiapoptotic and proapoptotic subfamilies [27]. Te combined treatment of PB and PF signifcantly decreased the expression of antiapoptotic proteins Bcl-2 and Bcl-xL and increased the expression of proapoptotic proteins Bax and Bad in Cal27 and Cal27/CDDP cells, compared with PB or PF treatment alone. Collectively, these results indicated that PB enhanced the intrinsic apoptosis-inducing efect of PF in TSCC cells.
Te PI3K/AKT/mTOR/p70S6K signaling pathway plays an important role in the occurrence and development of tumors. Jhou et al. [28] reported that chlorpromazine induced G 2 /M arrest and apoptosis via inhibiting the PI3K/ AKT/mTOR pathway in oral cancer. Results of a previous study demonstrated that activation of the PI3K/AKT/ mTOR/p70S6K signaling pathway is closely associated with chemoresistance in tumors. Lu et al. [29] found that the noncoding RNA-regulator of reprogramming (ROR) enhanced the sensitivity of breast cancer cells to the  chemotherapeutic drug tamoxifen through inhibition of the PI3K/AKT/mTOR signaling pathway. Li et al. [30] also demonstrated that the downregulation of the PI3K/AKT/ p70S6K signaling pathway using Guajadial reversed the multidrug resistance of breast cancer cells. Results of the present study demonstrated that the expression levels of PI3K and the ratios of phosphorylated (p)-AKT/AKT, p-mTOR/mTOR, and p-p70S6K/p70S6K were all decreased in the PB + PF group, compared with either of the PF or PB groups. Future studies will focus on the use of quantum nano-related technology to achieve targeted and efcient drug delivery. Collectively, the results of the present study demonstrated that PB synergistically enhanced the anticancer efect of PF chemotherapy. PB inhibited proliferation, invasion, and migration and induced apoptosis of TSCC cells by   regulating the PI3K/AKT/mTOR/p70S6K signaling pathways. Tis, therefore, enhanced the chemosensitivity of TSCC to PF.

Data Availability
Te datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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

Authors' Contributions
Shu-Ting Pan, Fang-Fei Ye, and Gan Huang contributed equally to this work. (f ) Ki67 expression in the PB + PF group was signifcantly lower than that in the control group. (g) H and E staining verifed that the PB + PF group did not induce toxicity in major organs (n � 6/group). PB, plumbagin; PF, cisplatin plus 5-fuorouracil; and TSCC, tongue squamous cell carcinoma.