Plumbagin

Plumbagin suppresses non-small cell lung cancer progression through downregulating ARF1 and by elevating CD8+ T cells

Ze-Bo Jiang a,b,1, Cong Xu a,b,1, Wenjun Wang a,b,1, Yi-Zhong Zhang a,b,1, Ju-Min Huang a,b, Ya-Jia Xie a,b, Qian-Qian Wang a,b, Xing-Xing Fan a,b, Xiao-Jun Yao a,b, Chun Xie a,b, Xuan-Run Wang a,b, Pei-Yu Yan a,b, Yu-Po Ma a,b,d,*, Qi-Biao Wu a,b,*, Elaine Lai-Han Leung a,b,c,*

A B S T R A C T

Non-small cell lung cancer (NSCLC) is one of the most frequently diagnosed cancers and the leading causes of cancer death worldwide. Therefore, new therapeutic agents are urgently needed to improve patient outcomes. Plumbagin (PLB), a natural sesquiterpene present in many Chinese herbal medicines, has been reported for its anti-cancer activity in various cancer cells. In this study, the effects and underlying mechanisms of PLB on the tumorigenesis of NSCLC were investigated. PLB dose-dependently inhibited the growth of NSCLC cell lines. PLB promoted ROS production, activated the endoplasmic reticulum (ER) stress pathway, and induced cell apoptosis, accompanied by the decreased expression level of ADP-ribosylation factor 1 (ARF1) in NSCLC cancer cells, and those effects of PLB could be reversed by the pretreatment with N-acetyl-L-cysteine (NAC). More importantly, the calcium chelator (BM) significantly reversed PLB-induced cell apoptosis. Furthermore, PLB significantly inhibited the growth of both H1975 xenograft and LLC1 tumors and exhibited antitumor activity by enhancing the number and the effector function of CD8+ T cells in KRASLA2 mice model and the LLC1 xenograft. Our findings suggest that PLB exerts potent antitumor activity against NSCLC in vitro and in vivo through ARF1 downregulation and induction of antitumor immune response, indicating that PLB is a new novel therapeutic candidate for the treatment of patients with NSCLC.

Keywords: Plumbagin ARF1 NSCLC ROS Immune response

1. Introduction

Lung cancer is one of the most frequently diagnosed cancers in men and women in the world [1,2]. Non-small cell lung cancer (NSCLC) is a clinically unique genomic subtype and immunologically heterogeneous disease [3]. Current treatments for NSCLC include surgery, radio- therapy, chemotherapy, targeted therapy, and immunotherapy, etc. [4–6]. Surgery is the best treatment option for NSCLC, but only approximately 25% of patients are suitable for potentially curative resection [7]. Chemotherapy is usually associated with numerous adverse reactions and severe side effects [8]. Although great progress has been made in the treatment strategies, such as targeted therapy and immunotherapy, the prognosis of lung cancer remains unsatisfactory [9]. Targeted therapy is one of the first-line therapies for NSCLC patients with activating mutations [10,11] and significantly improves their outcomes, but most patients treated with targeted therapy will eventu- ally develop drug resistance leading to therapeutic failure. New thera- peutic agents are urgently needed to overcome Tyrosine kinase inhibitor (TKI) resistance in NSCLC [8,12].
ADP-ribosylation factor 1 (ARF1) belongs to the Ras-related GTP binding proteins which are involved in the regulation of vesicular traf- ficking [13]. ARF1 plays a critically important role in cell signaling by cycling between the active state of GTP binding and the inactive state of GDP binding [14]. Overexpression of ARF1 is associated with poor prognosis in most cancers [15]. ARF1 ablation has an anti-cancer function by disrupting lipid metabolism in the mice model, resulting in the accumulation of lipid droplets, which further causes immune re- sponses to treat cancer in mice [16,17]. Ablation of ARF1 can induce endoplasmic ER stress response by driving mitochondrial defects in mice cancer stem cells and exhibits good antitumor activity by elevating DC cells and CD8+ T cells [17,18]. Plumbagin (PLB), a natural active sesquiterpene isolated from the traditional Chinese medicine (TCM) Plumbago zeylanica, has multiple biological activities against cancer cells in vitro and in vivo [19]. Increasing evidence has shown that PLB possesses various biological activities, such as inhibiting inflammatory responses, anti-bacterial, anti-cancer, and antioxidant properties, etc. [20–22]. PLB can also regulate lipid metabolism to improve obesity and non-alcoholic fatty liver [23]. Recently, there have been several studies attempting to investigate the effects of PLB on arthritis [24], obesity disease, cancer [25], and its immunosuppressive properties [26].
Nevertheless, the specific antitumor effects and underlying mecha- nisms of PLB in the treatment of NSCLC cancer cells have not been fully elucidated. In the present study, we provide evidence that PLB sup- pressed cell growth and promoted apoptosis in NSCLC cells with different mutations. Besides, PLB increased the intracellular ROS levels in H1975 and H1650 by targeting ARF1, induced the stress response of the endoplasmic reticulum, and elevated the level of intracellular cal- cium. More importantly, PLB also regulated antitumor effects and induced antitumor immune responses through ARF1. Plumbagin exhibited antitumor activity in KRASLA2 mice and induced an anti- tumor immune response.

2. Methods

2.1. Materials and Reagents

Plumbagin (Purity: ≥ 98%) was supplied by Cayman Chemicals (Ann Arbor, MI, USA). Primary antibodies against GAPDH (#5174S), PARP (#9532S), CHOP(#2895S), and anti-p-eIF2α(#3398S) were supplied by Cell Signaling Technology (Danvers, MA, USA). Fluo-3 AM (#ab145254), BAPTA-AM(#ab120503), and primary antibodies against ARF1(#ab11038) were supplied by Abcam (Cambridge, MA, USA). CM- H2DCFDA(#D399) was supplied by Thermofisher. Goat anti-rabbit and mouse secondary antibodies were supplied by Odyssey (Belfast, ME, USA). Annexin V-FITC/PI kit was supplied by BD Biosciences (San Jose, CA, USA). APC/Cyanine7 anti-mouse CD4 Antibody (#100414), PE/ Cyanine 7 anti-mouse CD8a Antibody (#100722), FITC anti-mouse CD3 Antibody (#100204), PE anti-mouse FOXP3 Antibody (#126404), PE anti-mouse IFN-γ Antibody (#505808), PE/Dazzle™ 594 anti-mouse TNF-α Antibody (#506346) and APC anti-human/mouse Granzyme B Recombinant Antibody (#396408) were supplied by BioLgend (San Diego, California, USA).

2.2. Cytotoxicity assay

Cells were respectively seeded into 96-well plates (5000 cells/ well) and were treated with a wide range of concentrations of PLB for 24 h. Then, the MTT reagent was added and incubated for 4 h. Finally, the colorimetric intensity of the plates was measured at the wave length of 570 nm by the Tecan microplate reader (Morrisville, NC, USA).

2.3. Colony formation assay

Colony formation assay was performed as described previously [27]. H1975 and H1650 cells were plated into 6 well plates and incubated overnight. Then the cells were treated with different concentrations of PLB for 24 h followed by two weeks to form colonies. When colony formation was visible, the medium was discharged. Then, colonies were washed with cold PBS, fixed with 4% paraformaldehyde (PFA) for at least 30 min, and then stained with 0.2% crystal violet solution in 10% ethanol for 20 min. The number of colonies formed of PLB on H1975, PC-9, H1650 and A549 was counted in each group by using the ste- reomicroscope. The picture of plates was captured by Epson perfection V700 photo.

2.4. Apoptosis detection

The percentage of Apoptosis cells treated with PLB was performed and analyzed as described previously [28]. Cells were resuspended and distributed in 6 well plates with 2 × 105 cells. After being treated with PLB for 24 h, the apoptotic cells were quantitatively determined by flow cytometry.

2.5. Mitochondrial membrane potential activity assay in NSCLC cancer

H1975 and H1650 cells were resuspended and distributed in 6 well plates with 2 × 105 cells and allowed to adhere overnight or 24 h. Briefly, the cells were treated with PLB for 24 h. After treatment for 24 h, cells were washed with ice-cold 1 ×PBS twice and digested with trypsin- EDTA. After centrifugation at 350 g for 5 mins, H1975 and H1650 cells were all collected. The cells were incubated with JC-1 for 30 mins in 5% CO2 incubator at 37 ◦C, subsequently harvested and washed with DPBS. Finally, all the samples were resuspended with DPBS, the red and green fluorescence was quantitatively analyzed by BD FACSaria 111 flow cy- tometer (BD Biosciences).

2.6. Detection of Ca2+ concentrations and calcium flux in NSCLC cancer

Changes in intracellular free calcium were measured as previously described [29]. Cells were harvested, washed twice, resuspended in Fluo-4/AM at 37◦C for 30 min, then were washed with PBS and analyzed by flow cytometry.

2.7. Measurement of reactive oxygen species generation of PLB

The cellular ROS content of PLB-treated NSCLC cell lines was measured as previously described [30]. Cells were stained with 10 µM DCFH-DA at 37 ◦C for 30 min and analyzed by FACSAria III flow cytometer (BD Biosciences).

2.8. Molecular docking analysis

The detailed procedure of molecular docking was as previously described [31]. The X-ray structure of ARF1 was retrieved from the protein database (PDB ID code1RE0) with a resolution of 2.40 Å for docking with PLB [32]. The molecular structure of ARF1 was prepared using standard procedure in the protein preparation wizard module of Schro¨dinger 2015. Using the Receptor Grid Generation tool in Glide, the docking grid frame was defined with the natural ligand in the ARF1 structure as the center. During the molecular docking process, the best binding of PLB was retained for further analysis.

2.9. Western blot analysis

The detailed procedure was as previously described [33]. The pri- mary antibodies were used as shown following: anti-p-eIF2α, CHOP, ARF1, ARG1GR and anti-eIF2α. The membranes were incubated with primary antibodies overnight at 4◦C. Then, the secondary fluorescent antibody (Odyssey) was added to the membrane at a 1:10,000 dilution at room temperature for 1 h. Protein expression was examined using an LI-COR Odyssey scanner (Belfast, ME, USA).

2.10. Transient transfection assay

The cells were seeded in 6-well dishes and grown to 70–80% confluence. Cells were transfected with lentiviral vectors to stably ex- press scrambled shRNA, ARF1 shRNA (Sigma-Aldrich, St. Louis, MO, USA). The concentrations of hygromycin or puromycin used for optimal selection of transfected cells depended on the type of cell lines. To determine the levels of Ca2+ and ROS, cells were washed in staining buffer and analyzed immediately using flow cytometry (BD Biosciences).

2.11. Mice model antitumor assay

All animal experiments were performed in accordance with the relevant guidelines and regulations approved by the Institutional Ani- mal Care and Use Committee. For the Lewis lung carcinoma model, mice were randomly divided into four experimental groups and treated with vehicle or (0.5, 1, 2 mg/ kg) of PLB. Lewis lung carcinoma model was treated for 21 days after tumor inoculation and the tumor weights were recorded. For H1975 xenograft mice model, female nude mice were injected subcutaneously with 5 × 105 H1975 cells, and then the dose 2 mg/kg of PLB was chosen for further studies. Mice were randomly divided into three experimental groups: control(Ctrl), PLB(2 mg/kg), and afatinib (10 mg/kg) group in which afatinib was used as a positive control.
The KRASLA2 mice were genotyped as described previously [34]. They were randomly assigned to 3 groups (n = 10) at the age of 5 weeks and treated daily with the vehicle and 2 mg/kg of PLB for 21 days. To analyzed the tumor multiplicity in the lungs, the tumor number in the lungs was calculated and recorded at the end of the experiment. The single-cell suspension cells were stained, acquired, and analyzed with the BD FACS Aria III flow cytometer.

2.12. Statistical analysis

All data were expressed as the mean ± SD of three individual ex- periments in this study. Differences between groups were determined by one-way analysis of variance (ANOVA), followed by the Bonferroni test to compare all pairs of columns. Survival rates of the Lewis lung carci- noma model were generated in accordance with the guidelines of the Kaplan–Meier method, statistical significance was determined based on the log-rank test. The result was considered to be statistically significant if P-value was < 0.05. 3. Results 3.1. PLB inhibited the growth and induced apoptosis in NSCLC cell lines To investigate the effects of PLB on NSCLC cell lines with different mutations, NSCLC cell lines were treated with PLB at a wide range of concentrations. Fig. 1A shows the chemical structure of PLB. As shown in Fig. 1B-C, PLB inhibited the growth of all NSCLC cell lines in a dose- dependent manner with a significant effect observed at 2–20 µM for up to 24 h. PLB also decreased the clonal growth in a dose-dependent manner (Fig. 1D-E). Next, we chose the H1975 and H1650 cell lines to analyze the apoptosis. H1975 is a NSCLC cell line harboring EGFR double mutations (L858R/T790M), and the H1650 NSCLC cell line also has the EGFR L858R mutation. As shown in Fig. 1F-G and Extended Data Fig. 1A–C, PLB induced apoptosis in a dose-dependent manner. Besides, we measured the mitochondrial membrane potential with JC-1 staining. The results demonstrated that PLB induced mitochondrial injury and thus inhibited the growth of NSCLC (Fig. 1H-I and Extended Data Fig. 1D-E). 3.2. Plumbagin upregulated apoptosis in NSCLC cells in an ARF1- dependent manner Guifang reported that ARF1 promoted cancer cell proliferation in ovarian cancer [35]. ARF1 could control the proliferation of breast cancer by regulating the retinoblastoma protein [14]. Yen designed and synthesized the ARF1-targeting compounds and revealed their strong cytotoxicity against HNSCC cells [36]. But so far, there is no research on the effect of Chinese medicine on ARF1 in lung cancer yet. Hence, we speculated that the mechanism of PLB in the treatment of NSCLC might be related to the inhibition of ARF1 expression in NSCLC cells. Molecular docking was calculated and performed the binding mode between PLB and ARF1. Fig. 2A–C showed that PLB could be bound and docked into ARF1 protein with a docking score of —8.948 ± 0.29 kcal/mol respectively. The hydrophobic region of ARF1, and experienced favorable hydrophobic and van der Waals interactions with residues TRP 78, MET 260, ASP 67, etc, which also facilitates the interaction between PLB and ARF1. Therefore, molecular docking data suggested that PLB could directly bind to the ARF1 protein. Then, the antitumor effects of PLB targeting ARF1 in H1975 and H1650 cells were investigated. PLB decreased ARF1 protein and mRNA levels in NSCLC cells (H1975 and H1650)(Fig. 2D-E). Extended Data Fig. 6A–C also demonstrated that ARF1 expression in the LLC1 cell line, H1975, and H1650 was successfully knocked down. In contrast to the above PLB inhibition experiments, PLB-induced apoptosis was signifi- cantly suppressed in H1650, H1975, and LLC cells with ARF1 knock- down (Fig. 2F–N). Finally, the MTT results also demonstrated that PLB suppresses H1975 and H1650 cell growth in an ARF1-dependent manner. 3.3. ARF1 regulated the plumbagin-induced oxidative stress and Ca2+ levels Fig. 3A–D showed that the levels of the Ca2+ levels and intracellular reactive oxygen species (ROS) were increased in both H1650 and H1975 cells after ARF1 was knocked down. ROS-induced apoptosis is reported to be one of the modes of action of the tumor-suppressing effect of plumbagin in other cancer cell lines [37]. Thus, the ROS levels were investigated with DCFDA. As shown in Fig. 3E–H, the ROS levels of H1975 and H1650 increased after treating with PLB. The ROS scavenger NAC was used to determine the role of ROS in inhibiting cancer for PLB. The MTT assay determined that NAC scavenger ROS significantly weakened the PLB-induced inhibition of cell proliferation in H1975 and H1650 (Fig. 3G-H). To further identify the role of ROS in PLB’s anti-cancer effects, as shown in Extended Data Fig. 2, pretreatment with NAC for 1 h markedly reversed the loss of mitochondrial membrane potential and increased apoptosis in NSCLC cell lines. Ca2+ elevation plays an important role in the anti-cancer activity and ER stress is accompanied by an alteration in the Ca2+ homeostasis, ER Ca2+ store depletion, and cell apoptosis. We then investigated whether PLB perturbed the intracellular Ca2+ homeostasis by using the flow cytometry technique. Fig. 3I-J showed that PLB increased Ca2+ levels in NSCLC cells. To ascertain whether Ca2+ elevation was associated with the PLB-induced NSCLC cell apoptosis, BAPTA/AM (BM), a Ca2+ chelator, was used in combination with PLB. The percentage of cells apoptotic significantly decreased after following the co-treatment with BM and PLB (Extended Data Fig. 3). Therefore, these data indicated that the PLB could increase the oxidative stress and Ca2+ level by reducing the ARF1 expression. Endoplasmic reticulum (ER) stress is becoming a modulator of various diseases and pathologies, and is an important mechanism for the death of cancer cells in response to different therapeutic agents [38]. Oxidative stress would occur together with the onset of ER stress in several anti-cancer instances [39,40]. Therefore, we used western blot to examine the protein expressions of ER stress-related, such as chop, ATF4, and p-eIF2αin PLB-treated cells. PLB treatment dose-dependently increased the expression of p-eIF2α and ATF4 in the H1975 and H1650 cells (Extended Data Fig. 3I-J). 3.4. Plumbagin exhibited antitumor activity through elevating CD8+ T cells in vivo Wang, et al. reported that ARF1 ablation in CT26 cells prevented tumor metastasis and induced antitumor immune response [17]. The correlation of cancer immune response and ARF1 in lung cancer was investigated in our study. LLC1 cells were knockdown for ARF1 expression using lentiviral vectors and utilized to induce subcutaneous tumor formation in female C57BL/6 mice. Extended Data Fig. 6 showed that the ablation of ARF1 suppressed cancer progression of LLC1 cells in vivo and in vitro. The single-cell suspensions of blood, spleen and tumor cells were stained and examined the antitumor effect of ARF1 inhibition in LLC cells on the production of TNFα and INF-γ by CD8+ T cells and the MHC-2 expression in DC cells. The above data verified that the ARF1 inhibition in LLC cells caused immune responses and suppressed NSCLC progression in vivo (Extended Data Figs. 4–6). The treatment of Lewis lung carcinoma mice with 2 mg/kg PLB improved the mice survival (Fig. 4A). The bodyweight of PLB-treated mice did not change compared to the untreated animals (Fig. 4B). Fig. 5C–E also showed that 2 mg/kg PLB suppressed LLC tumor growth starting at day 15 and showed a significant suppression from day 15 to day 18 while compared to the vehicle controls. We analyzed the changes in T cells and found that, compared with the vehicle control group, the number of CD8+ T cells increased with better activity in the PLB-treatment group (Fig. 4F and Extended Data Figs. 4–5). The number of tumor-infiltrating CD8+ T cells was increased in PLB-treated mice (Extended Data Fig. 4), and these cells showed po- tential effector function, CD8+ T cells showed a stronger activation phenotypes and enhanced the effector function of CD8+ T by increasing IFN-γ CD8+ T cells, TNFα CD8+ T cells, and Granzyme B (GrzmB) CD8+ T cells. Besides, the MHC-2 /DC cells in plumbagin-treated mice were increased in the tumor (Extended Data Fig. 4). 3.5. PLB suppressed the growth of the H1975 xenograft tumor in vivo The biological function of ARF1 in NSCLC is still unknown. We used the lentiviral vectors with control shRNA and ARF1shRNA to knock down ARF1 expression in H1975 and induced subcutaneous tumor formation in female nude mice. Fig. 5A–D showed that H1975 with knocked down ARF1 had a significantly smaller tumor size and volume compared with the control. Female H1975 xenograft mice were used to verify the antitumor ef- fect of PLB in vivo with the dose 2 mg/kg, afatinib-treatment was used as a positive control. PLB-treated mice did not show significant weight loss or apparent toxicity after treatment with plumbagin during the experi- mental period, but afatinib-treatment induced bodyweight loss (Fig. 5E). Fig. 4F showed that, compared to the vehicle control, 2 mg/kg plum- bagin suppressed H1975 tumor growth starting from day 15, and showed a significant suppression from day 15 to day 21. The tumor weights of the mice treated with 2 mg/kg plumbagin or afatinib were also significantly lower than those of the vehicle control group (Fig. 5G, H). 3.6. PLB exhibited antitumor activity in KRASLA2 mice Then, we investigated whether PLB had antitumor activity in KRASLA2 mice. The KRAS LA2 mice were divided into 2 groups (vehicle vs PLB) and treated with PLB (Fig. 6A). After treatment with PLB for 4 weeks, the mice were euthanized and the lung tumors were harvested for analysis. We found that PLB single treatment can inhibit tumor growth (Fig. 6B-C). We found PLB significantly increased CD8+ T cells and enhanced CD8+ T cells activation by increasing TNFα, IFN-γ, and Granzyme B expression in the PLB-treatment group (Fig. 6D–U). More interestingly, the number of CD4+ T cells was not changed by PLB treatment in the blood. However, the CD8/CD4 T cells ratio in spleen and blood increased, but the proportion of CD4+FOXP3+T cell in the spleen of PLB-treated mice decreased significantly (Fig. 6Q). 4. Discussion Lung cancer remains one of the most common cancers with an extremely poor prognosis [2,41], new therapeutic agents and more effective treatments are urgently needed to improve patient outcomes [1]. In this study, we reported that PLB, a natural active quinoid con- stituent isolated from the roots of the Chinese medicinal plant Plumbago zeylanica, effectively suppressed the proliferation and progression of NSCLC cells in vitro and in vivo. A few previous studies have reported that PLB has potent anticancer activity in some other cancer types, but its impact on NSCLC cells remains unclear [21,42]. Our study disclosed for the first time that PLB also had satisfying anticancer activity against NSCLC in vivo and in vitro. The analysis of immune subpopulations has shown that PLB treatment increased CD8+ infiltration T cells and exhibited good antitumor activity in KRAS LA2 mice. Besides, our xenograft mouse assay demonstrated that PLB (2 mg/kg, I.P.) did not show any apparent systemic toxicity. Until now, the mechanisms underlying the effects of PLB on cancer have not been fully understood [21,42], and the molecular/cellular targets of PLB in NSCLC cells remain unidentified. A previous study showed that PLB could increase the ROS production in prostate cancer PC cells [21]. Our study also indicated that PLB stimulated an increase in ROS production in NSCLC cancer cell H1975 and H1650 cells. ROS has various physiological response signal molecules, such as activate cell proliferation and survival pathways in some cells [43]. On the other hand, ROS may promote mutagenesis and suppress cancer cell pro- gression by damaging DNA when it moderately increases its level [44]. However, high ROS levels can induce oxidative stress on cells, which may eventually inhibit cell proliferation and cause cell senescence or death [45]. Therefore, it is possible that drugs may increase the level of ROS to selectively kill cancer cells [46]. PLB has been used safely in TCM for centuries to treat various diseases, including microbial infections [47]. Recently, it has been reported in the literature that PLB is a promising anticancer compound with multiple biological activities in some cancer models [48,49]. Increasing evidence shows PLB can increase ROS levels and induce oxidative stress to inhibit the proliferation of many tumors [21]. The present study indicated that PLB could increase ROS level and suppress the H1975 and H1650 cell proliferation. Calcium ion (Ca2+) is a critical second messenger in the cell and regulates various cellular biological functions including secretion, metabolism, cell proliferation, cell survival, cell cycle, cell death, and gene expression [50,51]. ROS communicates with several other systems and signaling pathways in biological activities, such as neurons, mito- chondria, which are also associated with calcium [52]. In the present study, PLB increased the ER stress regulator proteins expression by increasing the level of intracellular ROS and free calcium ions in H1975 and H1650 (Fig. 7). Interestingly, the PLB-induced cell apoptosis was significantly reversed by blockade of ROS production with the ROS inhibiter NAC. Compared with treatment with PLB, the mech- anism underlying NAC treatment is also associated with the induction of ER stress activation. In vivo xenograft tumor, beginning 15 days after ectopic implantation of H1975 cells, PLB reduced both tumor weight and tumor volume. ADP-ribosylation factors, which belong to the Ras-related GTP binding proteins, are well characterized as critical regulators for vesic- ular trafficking. ARF1 plays an important role as a molecule in cell signaling by cycling between the active state of GTP binding and the inactive state of GDP binding [17]. ARF1-ablation has an anti-cancer function by disrupting lipid metabolism in the mice model for results in the accumulation of lipid droplets, which further causes antitumor immune responses in mice [16,17]. ARF1 Ablation in mice cancer stem cells contributed to mitochondrial defects, thus causing endoplasmic ER stress response and exhibited antitumor activity by elevating CD8+ T cells [17]. In this paper, we confirm a potential novel mechanism of PLB that controls NSCLC cell line growth by ablation ARF1, increased ROS level, and increase Ca2+ levels. 5. Conclusions Plumbagin suppresses cell proliferation and improves the survival of NSCLC in vitro and in vivo by elevating CD8+ T cells and downregulation of the ARF1, which is a pivotal molecular target of plumbagin in NSCLC. Overall, our results suggest that plumbagin is a promising anticancer drug for the treatment of NSCLC. References [1] C. Wu, M. Li, H. Meng, Y. Liu, W. Niu, Y. Zhou, R. Zhao, Y. Duan, Z. Zeng, X. Li, G. Li, W. Xiong, M. Zhou, Analysis of status and countermeasures of cancer incidence and mortality in China, Sci. China Life Sci. 62 (5) (2019) 640–647. [2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2019, CA Cancer J. Clin. 69 (1) (2019) 7–34. [3] A. Singh, A. Daemen, D. Nickles, S.M. Jeon, O. Foreman, K. Sudini, F. Gnad, S. Lajoie, N. Gour, W. Mitzner, S. Chatterjee, E.J. Choi, B. Ravishankar, A. Rappaport, N.S. Patil, M. McCleland, L. Johnson, G.K. Acquaah-Mensah, E. Gabrielson, S. Biswal, G. Hatzivassiliou, NRF2 activation promotes aggressive lung cancer and associates with poor clinical outcomes, Clin. Cancer Res. (2020). [4] H. Sa, P. Song, K. Ma, Y. Gao, L. Zhang, D. Wang, Perioperative targeted therapy or immunotherapy in non-small-cell lung cancer, OncoTargets Ther. 12 (2019) 8151–8159. [5] N.E. Ready, P.A. Ott, M.D. Hellmann, J. Zugazagoitia, C.L. Hann, F. de Braud, S. J. Antonia, P.A. Ascierto, V. Moreno, A. Atmaca, S. Salvagni, M. Taylor, A. Amin, D. R. Camidge, L. Horn, E. Calvo, A. Li, W.H. Lin, M.K. Callahan, D.R. Spigel, Nivolumab monotherapy and nivolumab plus ipilimumab in recurrent small cell lung cancer: results from the CheckMate 032 randomized cohort, J. Thorac. Oncol. (2019). [6] C. Fang, C. Zhang, W.-Q. Zhao, W.-W. Hu, J. Wu, M. Ji, Co-mutations of TP53 and KRAS serve as potential biomarkers for immune checkpoint blockade in squamous- cell non-small cell lung cancer: a case report, BMC Med. Genom. 12 (1) (2019) 136. [7] B.S.L.A.Sa.L. Rydzewsk, A systematic review and meta-analysis of the literature: chemotherapy and surgery versus surgery alone in non-small cell lung cancer, J. Thorac. Oncol. 1.7 (2006) 611–621. [8] T.K. Tun AM, Thein WL, Guevara E., Checkpoint inhibitors plus chemotherapy for first-line treatment of advanced non-small cell lung cancer a systematic review and meta-analysis of randomized controlled trials.pdf>, ：Future Sci OA (2019).
[9] J. Malhotra, S.K. Jabbour, J. Aisner, Current state of immunotherapy for non-small cell lung cancer, Transl. Lung Cancer Res. 6 (2) (2017) 196–211.
[10] M. Yuan, L.L. Huang, J.H. Chen, J. Wu, Q. Xu, The emerging treatment landscape of targeted therapy in non-small-cell lung cancer, Signal Transduct. Target. Ther. 4 (2019) 61.
[11] P.R. Halliday, C.M. Blakely, T.G. Bivona, Emerging targeted therapies for the treatment of non-small cell lung cancer, Curr. Oncol. Rep. 21 (3) (2019) 21.
[12] X. Ai, X. Guo, J. Wang, A,L. Stancu, P.M.N. Joslin, D. Zhang, S. Zhu, Targeted therapies for advanced non-small cell lung cancer, Oncotarget 9 (101) (2018) 37589–37607.
[13] C. Casalou, A. Ferreira, D. Barral, The role of ARF family proteins and their regulators and effectors in cancer progression: a therapeutic perspective, Front. Cell Dev. Biol. 8 (2020) 217.
[14] P. Boulay, S. Schlienger, S. Lewis-Saravalli, N. Vitale, G. Ferbeyre, A. Claing, ARF1 controls proliferation of breast cancer cells by regulating the retinoblastoma protein, Oncogene 30 (36) (2011) 3846–3861.
[15] C. Casalou, A. Ferreira, D.C. Barral, The role of ARF family proteins and their regulators and effectors in cancer progression: a therapeutic perspective, Front. Cell Dev. Biol. 8 (2020) 217.
[16] C. D’Souza-Schorey, P. Chavrier, ARF proteins: roles in membrane traffic and beyond, Nat. Rev. Mol. Cell Biol. 7 (5) (2006) 347–358.
[17] G. Wang, J. Xu, J. Zhao, W. Yin, D. Liu, W. Chen, S.X. Hou, Arf1-mediated lipid metabolism sustains cancer cells and its ablation induces anti-tumor immune responses in mice, Nat. Commun. 11 (1) (2020) 220.
[18] J. Pencik, M. Schlederer, W. Gruber, C. Unger, S.M. Walker, A. Chalaris, I.J. Marie, M.R. Hassler, T. Javaheri, O. Aksoy, J.K. Blayney, N. Prutsch, A. Skucha, M. Herac, O.H. Kramer, P. Mazal, F. Grebien, G. Egger, V. Poli, W. Mikulits, R. Eferl, H. Esterbauer, R. Kennedy, F. Fend, M. Scharpf, M. Braun, S. Perner, D.E. Levy, T. Malcolm, S.D. Turner, A. Haitel, M. Susani, A. Moazzami, S. Rose-John, F. Aberger, O. Merkel, R. Moriggl, Z. Culig, H. Dolznig, L. Kenner, STAT3 regulated ARF expression suppresses prostate cancer metastasis, Nat. Commun. 6 (2015) 7736.
[19] T. Li, M. Lv, X. Chen, Y. Yu, G. Zang, Z. Tang, Plumbagin inhibits proliferation and induces apoptosis of hepatocellular carcinoma by downregulating the expression of SIVA, Drug Des. Dev. Ther. 13 (2019) 1289–1300.
[20] Y. Chen, C. Zhao, X. Liu, G. Wu, J. Zhong, T. Zhao, J. Li, Y. Lin, Y. Zhou, Y. Wei, Plumbagin ameliorates liver fibrosis via a ROS-mediated NF-small ka, CyrillicB signaling pathway in vitro and in vivo, Biomed. Pharm. 116 (2019), 108923.
[21] H. Huang, H. Xie, Y. Pan, K. Zheng, Y. Xia, W. Chen, Plumbagin triggers ER stress- mediated apoptosis in prostate cancer cells via induction of ROS, Cell. Physiol. Biochem. 45 (1) (2018) 267–280.
[22] X. Kong, J. Luo, T. Xu, Y. Zhou, Z. Pan, Y. Xie, L. Zhao, Y. Lu, X. Han, Z. Li, L. Liu, Plumbagin enhances TRAIL-induced apoptosis of human leukemic Kasumi1 cells through upregulation of TRAIL death receptor expression, activation of caspase-8 and inhibition of cFLIP, Oncol. Rep. 37 (6) (2017) 3423–3432.
[23] S.A. Pai, R.P. Munshi, F.H. Panchal, I.S. Gaur, S.N. Mestry, M.S. Gursahani, A. R. Juvekar, Plumbagin reduces obesity and nonalcoholic fatty liver disease induced by fructose in rats through regulation of lipid metabolism, inflammation and oxidative stress, Biomed. Pharm. 111 (2019) 686–694.
[24] T. Wang, H. Qiao, Z. Zhai, J. Zhang, J. Tu, X. Zheng, N. Qian, H. Zhou, E. Lu, T. Tang, Plumbagin ameliorates collagen-induced arthritis by regulating Treg/ Th17 cell imbalances and suppressing osteoclastogenesis, Front. Immunol. 9 (2018) 3102.
[25] S.S. Messeha, N.O. Zarmouh, P. Mendonca, H. Alwagdani, M.G. Kolta, K.F. A. Soliman, The inhibitory effects of plumbagin on the NF-B pathway and CCL2 release in racially different triple-negative breast cancer cells, PLoS One 13 (7) (2018), 0201116.
[26] W. Kuan-Hong, L. Bai-Zhou, Plumbagin protects against hydrogen peroxide- induced neurotoxicity by modulating NF-kappaB and Nrf-2, Arch. Med. Sci. 14 (5) (2018) 1112–1118.
[27] U. De, J.Y. Son, Y. Jeon, S.-Y. Ha, Y.J. Park, S. Yoon, K.-T. Ha, W.S. Choi, B.M. Lee, I.S. Kim, J.H. Kwak, H.S. Kim, Plumbagin from a tropical pitcher plant (Nepenthes alata Blanco) induces apoptotic cell death via a p53-dependent pathway in MCF-7 human breast cancer cells, Food Chem. Toxicol. 123 (2019) 492–500.
[28] X. Li, X.X. Fan, Z.B. Jiang, W.T. Loo, X.J. Yao, E.L. Leung, L.W. Chow, L. Liu, Shikonin inhibits gefitinib-resistant non-small cell lung cancer by inhibiting TrxR and activating the EGFR proteasomal degradation pathway, Pharm. Res. 115 (2017) 45–55.
[29] W. Wang, L. Mao, H. Lai, Y. Wang, Z. Jiang, W. Li, J. Huang, Y. Xie, C. Xu, P. Liu, Y. Li, E. Leung, X. Yao, Dolutegravir derivative inhibits proliferation and induces apoptosis of non-small cell lung cancer cells via calcium signaling pathway, Pharmacol. Res. 161 (2020), 105129.
[30] F. Duan, M. Wang, Y. Cao, Dan Li, R. Li, X. Fan, I. Khan, H. Lai, Y. Zhang, W. Hsiao, X. Yao, Q. Wu, L. Liu, Y. Tang, E. Leung, MicroRNA-421 confers paclitaxel resistance by binding to the KEAP1 3′UTR and predicts poor survival in non-small cell lung cancer, Cell Death Dis. 10 (11) (2019) 821.
[31] W.J. Wang, L.F. Mao, H.L. Lai, Y.W. Wang, Z.B. Jiang, W. Li, J.M. Huang, Y.J. Xie, C. Xu, P. Liu, Y.M. Li, E.L.H. Leung, X.J. Yao, Dolutegravir derivative inhibits proliferation and induces apoptosis of non-small cell lung cancer cells via calcium signaling pathway, Pharm. Res. 161 (2020), 105129.
[32] E. Mossessova, R.A. Corpina, J. Goldberg, Crystal structure of ARF1• Sec7 complexed with Brefeldin A and its implications for the guanine nucleotide exchange mechanism, Mol. Cell 12 (6) (2003) 1403–1411.
[33] Z. Jiang, J. Huang, Y. Xie, Y. Zhang, C. Chang, H. Lai, W. Wang, X. Yao, X. Fan, Q. Wu, C. Xie, M. Wang, E. Leung, Evodiamine suppresses non-small cell lung cancer by elevating CD8 T cells and downregulating the MUC1-C/PD-L1 axis, J. Exp. Clin. Cancer Res. CR 39 (1) (2020) 249.
[34] E.L.-H. Leung, L.X. Luo, Y. Li, Z.-Q. Liu, L.L. Li, D.F. Shi, Y. Xie, M. Huang, L.L. Lu, F.G. Duan, J.M. Huang, X.X. Fan, Z.W. Yuan, J. Ding, X.J. Yao, D.C. Ward, L. Liu, Identification of a new inhibitor of KRAS-PDEδ interaction targeting KRAS mutant nonsmall cell lung cancer, Int. J. Cancer 145 (2019) 1334–1345.
[35] Guifang Gu, Yanna Chen, Chengwei Duan, Linsen Zhou, Caiwen Jie, Overexpression of ARF1 is associated with cell proliferation and migration through PI3K signal pathway in ovarian cancer, Oncol. Rep. (2017).
[36] Y. Vo-Hoang, S. Paiva, L. He, S. Estaran, Y. Teng, Design and synthesis of Arf1- targeting γ-dipeptides as potential agents against head and neck squamous cell carcinoma, Cells 9 (2) (2020) 286.
[37] W. Chen, P. Li, Y. Liu, Y. Yang, X. Ye, F. Zhang, H. Huang, Isoalantolactone induces apoptosis through ROS-mediated ER stress and inhibition of STAT3 in prostate cancer cells, J. Exp. Clin. Cancer Res. 37 (1) (2018) 309.
[38] P. Limonta, R. Moretti, M. Marzagalli, F. Fontana, M. Raimondi, M. Montagnani Marelli, Role of endoplasmic reticulum stress in the anticancer activity of natural compounds, Int. J. Mol. Sci. 20 (4) (2019).
[39] Z. Zhang, L. Zhang, L. Zhou, Y. Lei, Y. Zhang, C. Huang, Redox signaling and unfolded protein response coordinate cell fate decisions under ER stress, Redox Biol. 25 (2019), 101047.
[40] W. Chen, P. Li, Y. Liu, Y. Yang, X. Ye, F. Zhang, H. Huang, Isoalantolactone induces apoptosis through ROS-mediated ER stress and inhibition of STAT3 in prostate cancer cells, J. Exp. Clin. Cancer Res. CR 37 (1) (2018) 309.
[41] W. Chen, R. Zheng, P.D. Baade, S. Zhang, H. Zeng, F. Bray, A. Jemal, X.Q. Yu, J. He, Cancer statistics in China, 2015, CA Cancer J. Clin. 66 (2) (2016) 115–132.
[42] Y.Y. Cao, J. Yu, T.T. Liu, K.X. Yang, L.Y. Yang, Q. Chen, F. Shi, J.J. Hao, Y. Cai, M. R. Wang, W.H. Lu, Y. Zhang, Plumbagin inhibits the proliferation and survival of esophageal cancer cells by blocking STAT3-PLK1-AKT signaling, Cell Death Dis. 9 (2) (2018) 17.
[43] J. Roy, J. Galano, T. Durand, J. Le Guennec, J. Lee, Physiological role of reactive oxygen species as promoters of natural defenses, FASEB J. Off. Publ. Fed. Am. Soc. Exp. Biol. 31 (9) (2017) 3729–3745.
[44] M. Schieber, N.S. Chandel, ROS function in redox signaling and oxidative stress, Curr. Biol. 24 (10) (2014) R453–R462.
[45] N.P. Degtyareva, L. Heyburn, J. Sterling, M.A. Resnick, D.A. Gordenin, P. W. Doetsch, Oxidative stress-induced mutagenesis in single-strand DNA occurs primarily at cytosines and is DNA polymerase zeta-dependent only for adenines and guanines, Nucleic Acids Res. 41 (19) (2013) 8995–9005.
[46] E.L. Leung, X.X. Fan, M.P. Wong, Z.H. Jiang, Z.Q. Liu, X.J. Yao, L.L. Lu, Y.L. Zhou, L.F. Yau, V.P. Tin, L. Liu, Targeting Tyrosine Kinase Inhibitor-Resistant Non-Small Cell Lung Cancer by Inducing Epidermal Growth Factor Receptor Degradation via Methionine 790 Oxidation, Antioxid. Redox Signal. 24 (5) (2016) 263–279.
[47] S.V. Nair, G. Baranwal, M. Chatterjee, A. Sachu, A.K. Vasudevan, C. Bose, A. Banerji, R. Biswas, Antimicrobial activity of plumbagin, a naturally occurring naphthoquinone from Plumbago rosea, against Staphylococcus aureus and Candida albicans, Int. J. Med. Microbiol. 306 (4) (2016) 237–248.
[48] A. Kawiak, A. Domachowska, E. Lojkowska, Plumbagin increases paclitaxel- induced cell death and overcomes paclitaxel resistance in breast cancer cells through ERK-mediated apoptosis induction, J. Nat. Prod. 82 (4) (2019) 878–885.
[49] M.H. Aziz, N.E. Dreckschmidt, A.K. Verma, Plumbagin, a medicinal plant–derived naphthoquinone, is a novel inhibitor of the growth and invasion of hormone- refractory prostate cancer, Cancer Res. 68 (21) (2008) 9024–9032.
[50] A. Gorlach, K. Bertram, S. Hudecova, O. Krizanova, Calcium and ROS: a mutual interplay, Redox Biol. 6 (2015) 260–271.
[51] E. Varghese, S.M. Samuel, Z. Sadiq, P. Kubatka, A. Liskova, J. Benacka, P. Pazinka, P. Kruzliak, D. Busselberg, Anti-cancer agents in proliferation and cell death: the calcium connection, Int. J. Mol. Sci. 20 (12) (2019).
[52] S. Feno, G. Butera, D. Vecellio Reane, R. Rizzuto, A. Raffaello, Crosstalk between calcium and ROS in pathophysiological conditions, Oxid. Med. Cell Longev. 2019 (2019), 9324018.