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. 2023 Aug 5;46(6):1837–1853. doi: 10.1007/s13402-023-00850-5

Survivin degradation by bergenin overcomes pemetrexed resistance

Xiaoying Li 1,2,#, Qi Liang 1,2,#, Li Zhou 3,#, Gaoyan Deng 4, Yeqing Xiao 5, Yu Gan 1,2, Shuangze Han 1,6, Jinzhuang Liao 1,2, Ruirui Wang 1,2, Xiang Qing 7,✉, Wei Li 1,2,✉
PMCID: PMC12974698  PMID: 37542022

Abstract

Purpose

Chemoresistance is a primary factor for treatment failure and tumor recurrence in non-small cell lung cancer (NSCLC) patients. The oncoprotein survivin is commonly upregulated in human malignancies and is associated with poor prognosis, but its effect on carcinogenesis and chemoresistance in NSCLC is not yet evident, and to explore an effective inhibitor targeting survivin expression is urgently needed.

Methods

The protumor characteristics of survivin and antitumor activities of bergenin in NSCLC cells were examined by MTS, colony formation assays, immunoblot, immunohistochemistry, and in vivo xenograft development.

Results

Survivin was upregulated in non-small cell lung cancer (NSCLC) tissues, while its depletion inhibited NSCLC tumorigenesis. The current study focused on bergenin, identifying its effective antitumor effect on NSCLC cells both in vivo and in vitro. The results showed that bergenin could inhibit cell proliferation and induce the intrinsic pathway of apoptosis via downregulating survivin. Mechanistically, bergenin reduced the phosphorylation of survivin via inhibiting the Akt/Wee1/CDK1 signaling pathway, thus resulting in enhanced interaction between survivin and E3 ligase Fbxl7 to promote survivin ubiquitination and degradation. Furthermore, bergenin promoted chemoresistance in NSCLC cells re-sensitized to pemetrexed treatment.

Conclusions

Survivin overexpression is required for maintaining multiple malignant phenotypes of NSCLC cells. Bergenin exerts a potent antitumor effect on NSCLC via targeting survivin, rendering it a promising agent for the treatment of NSCLC.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13402-023-00850-5.

Keywords: Non-small cell lung cancer, Survivin, Bergenin, Ubiquitination, Chemoresistance

Introduction

Non-small cell lung cancer (NSCLC) is the predominant subtype of lung cancer, accounting for 80–85% of all lung cancer cases, and remains the primary cause of cancer-related mortality worldwide [2, 43]. NSCLC is subdivided into adenocarcinoma, squamous cell carcinoma, large cell carcinoma, and mixed histology [44]. Despite recent advances in the diagnosis and treatment modalities of NSCLC, only a small proportion of patients (< 20%) are diagnosed early due to the lack of particular symptoms, and most (47%) are diagnosed at an advanced stage [42]. The therapeutic strategies for treating NSCLC include traditional surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy [12, 30]. Although monotherapy and combination therapy have improved the quality of life and overall survival of patients, tumor progression and treatment resistance are still major impediments to overcome [5, 8, 21, 28, 41]. Therefore, advancing pre/clinical trials for developing efficacious agents to improve the management of NSCLC is of great importance.

Survivin, a member of the inhibitors of apoptosis proteins (IAPs) with numerous functions, plays a crucial role in regulating cell division and apoptosis. It is located in different subcellular components, such as the nucleus, cytoplasm, mitochondria, and extracellular space. Nuclear survivin is involved in the formation of the chromosome passenger complex and regulates cell division. On the other hand, the cytoplasmic and mitochondrial expression of survivin is associated with the suppression of apoptosis [10, 22]. Tumor cells are considered as the extracellular pool of survivin via secreting survivin in the form of membrane vesicles that are absorbed by surrounding cells. The above process can promote therapy resistance and tumor development [20]. Accumulating evidences has suggested that survivin is highly expressed in embryonic tissues and cancer cells but rarely in normal adult tissues [15]. The expression of survivin is closely associated with cell proliferation, invasion and metastasis, recurrence, therapeutic resistance and poor prognosis in several types of cancer, including lung cancer, breast cancer, ovarian cancer and gastric cancer [13, 34, 45, 57], thus providing a potential target for improving the treatment of patients with NSCLC.

Compared with traditional chemotherapeutic drugs, natural products have gradually become attractive therapeutic agents due to their less cytotoxic and side effects [59]. Bergenin, a C-glucoside of 4-O-methylgallic acid, is an active constituent of the plants of the genus bergenia, with a wide range of pharmacological activities, including anti-arrhythmic, anti-inflammatory, hepatoprotective, neuroprotective and antitumor properties [29, 38, 55, 56]. Previous studies showed that bergenin could attenuate bleomycin-induced pulmonary fibrosis via inhibiting the TGF-β1 signaling [25, 27], ameliorate cognitive deficit attributed to its regulatory effect on oxidative stress and neuroprotective action [40] and protect against acute lung injury by inhibiting NF-κB activation [52]. Currently, the pharmacological activity of bergenin has been mainly investigated in non-neoplastic diseases, while its inhibitory effect on malignant tumors has rarely been reported. The present study demonstrated that bergenin exerted strong antitumor activity against NSCLC cells both in vitro and in vivo, and the underlying mechanism of this pharmacological function was investigated.

Materials and methods

Cell culture and reagents

The immortalized lung epithelial cell lines NL20 and MRC5, and human NSCLC A549, H1299 and H460 cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). All cells were grown in a humidified incubator at 37℃ with 5% CO2 and subjected to mycoplasma analysis routinely. The cell culture medium of RPMI-1640 and Fetal Bovine Serum (FBS) were obtained from Invitrogen (Grand Island, NY). The natural products bergenin, MG132, cycloheximide (CHX), and pemetrexed (PEM) were obtained from Selleck Chemicals (Houston, TX). SDS, DMSO, NaCl, and Trisbase for buffer preparation and molecular biology were purchased from Sigma-Aldrich (Merck KGaA; St. Louis, MO, USA). Transfection reagent lipofectamine 2000 was purchased from Thermo Fisher Scientific (Waltham, MA). Antibodies against survivin (#2808), cleaved-PARP (#5625), cleaved-caspase 3 (#9664), VDAC1 (#4661), Bax (#5023), cytochrome C (#11940), p-CDK1 Thr161 (#9114), p-CDK1 Tyr15 (#4539), p-Wee1 Ser642 (#4910), p-Akt Ser473 (#4060), p-survivin Thr34 (#8888), ubiquitin (#3936), HA-tag (#3724), Flag-tag (#8146), α-Tubulin (#3873) and β-actin (#4970) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). The FbxL7 (#ab59149) and ki67 (#ab16667) antibodies were obtained from Abcam (Cambridge, United Kingdom).

Natural compound screening

A customized chemical library containing 303 compounds was purchased from MedChemExpress. A549 cells were seeded in a 96-well plate. After overnight incubation, cells were treated with 1 μM of DMSO (control) or natural compounds for 48 h. Cell viability was examined by the MTS assay. Tested compounds are listed in Supplementary Table S1.

MTS assay

The MTS assay, used to analyze cell viability, was performed as described previously [26]. Human NSCLC cells were seeded into the 96-well plates (2 × 103 cells/well) and maintained overnight. Cells were incubated with various concentrations of bergenin or PEM (1 µM) at different times. Then cell viability was analyzed according to the standard protocol after adding the MTS reagent (#G3580, Madison, WI) to the cell culture medium.

Anchorage-independent cell growth assay

The Anchorage-independent cell growth assay was performed as described previously [25, 27]. Briefly, Eagle’s basal medium consists of 0.6% agar, 10% FBS and various concentration of bergenin or PEM (1 µM), which were loaded in a 6-well plate as an agar base. Human NSCLC cells were resuspended at a concentration of 8000 cells/ml and inoculated in 6-well plates containing 0.6% Basal Medium Eagle (0.3% agar, 10% FBS, and different doses of bergenin. Colonies were counted after maintaining for 2 weeks.

Clinical tissue sample collections

A total of 40 cases of NSCLC tissues and matched adjacent non-tumor tissues were collected from 40 patients in the department of pathology, The Third Xiangya Hospital of Central South University, Changsha, Hunan, China. All the patients signed written informed consent and did not receive treatment before surgery. The study was reviewed and approved by the Ethics Committee of the Third Xiangya Hospital of Central South University (Changsha, China).

Western blotting

The western blotting was performed as formerly described [53]. The cells were treated with bergenin and lysed in RIRA buffer (#89901, Thermo Fisher Scientific) at 4℃ for around 30 min followed by centrifugation at 12,000 rpm for 10 min to collect supernatant as the whole-cell extract (WCE). Then the BCA protein assay kit (#23228, Thermo Fisher Scientific) was used to detect the WCE concentration. A total of 20 μg WCE was subjected to SDS-PAGE gels and transferred to the PVDF membrane. Subsequently, the membranes are blocked with 5% non-fat milk for 1 h and incubated with the primary and second antibodies. The ECL reagent (#34579, Thermo Fisher Scientific) was used to visualize the target protein expression.

Co-immunoprecipitation (Co-IP)

The Co-immunoprecipitation assay was performed as described previously [23]. Total proteins from NSCLC cells cultured in 100 mm culture dishes were used for the Co-IP assay. Briefly, primary antibodies against HA or Flag were added to each cell lysate and incubated overnight at 4℃ with rotation. Protein A/G agarose beads were added and incubated with mild rotation at 4 °C for 2 h to bind to the primary antibody. The agarose beads were washed with PBS and cell lysis buffer to wash off unbound antibodies. The agarose beads were resuspended in 20 μl 1xSDS-PAGE loading buffer and boiled and centrifuged to collect the supernatant for western blot analysis.

Generation of survivin knockdown stable cell lines

Gene knockdown stable cell lines were generated as described previously [58]. The 293 T cells were co-transfected with pLKO.1-shSurvivin lentivirus plasmids (TRCN0000073718, TRCN0000073721, Millipore Sigma), PSPAX2 and PMD2-G. The supernatant containing viral was collected at 72 h after transfection. NSCLC cells were grown at 70%-80% confluence and infected with the lentivirus and polybrene (5 µg/ml). The infected cells were cultured with fresh medium containing puromycin (1 μg/ml) and maintained for 1 week for colony selection.

In vivo tumor growth

The in vivo tumorigenesis was approved by the Institutional Animal Care and Use Committee (IACUC) of the Third Xiangya Hospital of Central South University (Changsha, China). The parental or PEM-resistant cell lines A549 and H1299 (2 × 106) were injected into the right flank of 6-week-old athymic nude mice (n = 5, Table S2). Tumor growth was monitored, and volume was measured every 2 days. The tumor-bearing mice were randomly divided into two groups (n = 5) when tumor volume reached around 100 mm3. The treatment group initiated bergenin (30 mg/kg) or PEM (3 mg/kg) via intraperitoneal injection every two days, and the control group was administered the vehicle control. For combined treatment, the tumor-bearing mice were randomly divided into four groups (n = 5, Table S2): 1, vehicle control (0.5% dimethyl sulfoxide in 100 µL Corn oil /every 2 days, i.p.); 2, bergenin (30 mg/kg/ in 100 µL Corn oil every 2 days, i.p.); 3, PEM (3 mg/kg/ in 100 µL Corn oil every 2 days, i.p.); 4, bergenin (30 mg/kg/ every 2 days, i.p.) + PEM (3 mg/kg/ every 2 days, i.p.).The mice were euthanized with CO2 (3 L/min) for 5 min at the endpoint. Tumor volume was calculated following the formula: length x width x width/2.

Immunohistochemical staining (IHC)

The IHC staining was performed as described previously [53]. Tumor tissues obtained from clinical samples and xenograft tumors were subjected to IHC analysis. Briefly, the tissue sections were deparaffinized and rehydrated. The antigen retrieval was performed subsequently by submerging into sodium citrate buffer (10 mM, pH 6.0) and boiling for 10 min. Then the tissue slides were treated with 3% H2O2 in methanol for 10 min, washed with PBS and blocked with 10% goat serum albumin, and incubated with primary antibodies overnight at 4℃. After hybridization with secondary antibodies at room temperature, the target protein was visualized using the DAB substrate and counterstained by hematoxylin.

Statistical analysis

GraphPad Prism 5 (GraphPad 5.0, San Diego, CA, USA) software was used for statistical analysis. Data were performed from at least three independent determinations and represented as mean ± sd. Statistical comparisons between different groups were analyzed by Student's t-test or ANOVA. A probability value of p < 0.05 was used as the criterion for statistical significance (*p < 0.05, **p < 0.01, ***p < 0.001).

Results

Survivin is overexpressed in NSCLC tumor tissues

The expression levels of survivin in tumor and adjacent non-tumorous tissues collected from 40 patients with NSCLC were determined by IHC staining. The results showed that survivin expression in tumor tissues is much higher than that in adjacent tissues (Fig. 1A). The Kaplan-Meier survival analysis revealed that the increased survivin expression levels were associated with worse overall survival (OS), first progression survival (FP) and post-progression survival (PPS) in patients with NSCLC (Fig. 1B). To further evaluate its oncogenic role, survivin knockdown stable cell lines in A549 and H1299 cells were generated. MTS and anchorage-independent cell growth assay showed that depletion of survivin significantly inhibited the proliferation and metastasis potential of NSCLC cells (Fig. 1C and D). Moreover, the in vitro observations were verified in A549-derived and H1299-derived xenograft models, in which survivin knockdown significantly decreased tumor volume (Fig. 1E and S1A-C) and weight (Fig. 1F and S1D) and reduced the percentage of Ki67 positive cells (Fig. 1G). These results indicated that survivin was highly expressed in NSCLC tissues and was closely associated with the carcinogenic phenotype of NSCLC cells.

Fig. 1.

Fig. 1

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Knockdown of survivin inhibits the malignant phenotype of NSCLC cells. A IHC staining analysis of survivin in 40 cases of NSCLC tissues and matched adjacent non-tumor tissues. Left, The representative images of survivin IHC staining. Right, Qualification. Scale bar, 40 μm. ***p < 0.001. B Kaplan-Meier survival analysis for the relationship between survival time and survivin signature in NSCLC was performed using the online tool (http://kmplot.com/analysis/). OS (left), Overall Survival. FP (middle), First Progression Survival. PPS (right), Post Progression Survival. C Cell viability of A549 (left) and H1299 (right) cells expressing shCtrl or shsurvivin. Top, IB analysis of survivin protein level. Bottom, MTS analysis of cell viability. ***p < 0.001. D The colony formation of A549 and H1299 cells expressing shCtrl or shsurvivin. ***p < 0.001. E–G In vivo tumorigenesis of A549 expressing shCtrl or shsurvivin. Tumor volume (E), tumor weight (F), and IHC staining analysis of the population of Ki67 positive cells (G) of the A549 xenografts (n = 5). Scale bar, 25 μm. ***p < 0.001. Data were performed from at least three independent determinations and represented as mean ± sd

Bergenin exerts antitumor effects and facilitates Fbxl7-mediated survivin ubiquitination in NSCLC cells

To identify natural products that inhibit the growth of NSCLC cells, a customized natural compound library containing 303 compounds was screened using the MTS assay (Supplementary Table S1). It was found that bergenin reduced the viability of A549 cells by approximately 30% at a dosage of 20 μM (Fig. 2A). Subsequently, to verify the cytotoxic effect of bergenin on NSCLC cells (Fig. 2B), the immortalized non-tumorous lung tissue cell lines NL20 and MRC5 were firstly treated with bergenin. The results showed that cell viability was not significantly affected by bergenin treatment even at a concentration of 160 μM (Fig. 2C). Then, the half maximal inhibitory concentration of bergenin in NSCLC cell lines A549, H1299 and H460 was detected by MTS assay, and the results suggested that the IC50 values of bergenin in A549 cells was 42.55 μM, in H1299 cells was 21.89 μM, and in H460 cells was 10.17 μM (Fig. S2). Notably, following treatment with bergenin, the viability of A549, H1299 and H460 cells was markedly reduced in a dose-dependent manner (Fig. 2D). Consistently, the cologenesis experiment showed that bergenin could significantly impair the colony formation ability of NSCLC cells on a plate or in the soft agar dose-dependently (Fig. 2E and F). These results suggested that bergenin exhibited antitumor activity against NSCLC cells and was well-tolerated by immortalized non-tumorous cells.

Fig. 2.

Fig. 2

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Bergenin suppresses NSCLC cells. A A549 cells were treated with the screened compounds (20 μM) for 48 h. MTS assay was used to determine cell viability. Red dot, bergenin. B The chemical structure of bergenin. C MTS assay analysis of the cell viability of NL20 and MRC5 cells with bergenin treatment for 48 h. D MTS assay analysis of the cell viability of A549 (left), H1299 (middle), and H460 (right) cells with bergenin treatment for 48 h. *p < 0.05, **p < 0.01, ***p < 0.001. E Soft agar assay was employed to assess the anchorage-independent cell proliferation of A549 (up), H1299 (middle), and H460 (down) cells with bergenin treatment for 48 h. Scale bar, 500 μm. ***p < 0.001. F Plate colony formation assay was performed to analyze the colony formation of A549 (left), H1299 (middle), and H460 (right) cells with bergenin treatment for 48 h. ***p < 0.001. Data were performed from at least three independent determinations and represented as mean ± sd

To uncover the underlying antitumor mechanism of bergenin, the immunoblotting (IB) assay was performed. We found hat the expression of survivin was downregulated in A549 and H1299 cells treated with bergenin in a dose-dependent manner (Fig. 3A). The above effect was partially restored in a time-dependent manner following cell treatment with MG132, a proteasome inhibitor (Fig. 3B and C). Additionally, the half-life of survivin was shortened after treatment with bergenin, as demonstrated by the cycloheximide chase assay (Fig. 3D), indicating that the bergenin-induced survivin downregulation could be attributed to interference with its protein stability. The ubiquitination of survivin was then determined, and it was found that the ubiquitination of survivin was strongly enhanced in the presence of bergenin (Fig. 3E). Further detailed studies revealed that bergenin could enhance the interaction between survivin and the E3 ligase Fbxl7, resulting in upregulation of survivin ubiquitination (Fig. 3F). To determine whether E3 ligase Fbxl7 was indispensable for survivin ubiquitination in bergenin-treated NSCLC cells, A549 cells were transfected with small interfering (si)RNA sequences targeting Fbxl7 to silence its expression. The results demonstrated that Fbxl7 knockdown impaired bergenin-induced survivin ubiquitination (Fig. 3G). Furthermore, double mutation at the K90/91 residues, two ubiquitination sites required for survivin degradation, attenuated the Fbxl7-mediated survivin ubiquitination in A549 cells (Fig. 3H). Accordingly, the effect of bergenin on survivin ubiquitination was abrogated following cell transfection with Flag-survivin K90/91R (Fig. 3I). Overall, the above results indicated that bergenin could promote the ubiquitination and degradation of survivin via enhancing its interaction with Fbxl7.

Fig. 3.

Fig. 3

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Bergenin promotes ubiquitination-mediated survivin degradation. A A549 and H1299 cells were treated with bergenin for 48 h. The WCE was subjected to IB analysis. B A549 and H1299 cells were treated with bergenin for 48 h, and incubated with MG132 (20 µM) for 10 h. The WCE was subjected to IB analysis. C A549 and H1299 cells were treated with bergenin for 48 h, and incubated with MG132 (20 µM) for various time points. The WCE was subjected to IB analysis. D A549 cells were treated with bergenin for 48 h, and incubated with CHX for various time points. The WCE was subjected to IB analysis. E A549 cells were treated with bergenin for 48 h, and incubated with MG132 (20 µM) for 10 h. The WCE was subjected to survivin ubiquitination analysis. F A549 cells were transfected with HA-Fbxl7 and Flag-Survivin for 24 h, followed by bergenin treated for 48 h, and incubated with MG132 (20 µM) for 10 h. The WCE was subjected to co-IP analysis. G A549 cells were transfected with siFbxl7 for 24 h, treated with Bergenin for 48 h and incubated with MG132 (20 µM) for 10 h. The WCE was subjected to survivin ubiquitination analysis. H A549 cells were transfected with Flag-Survivin-WT or Flag-Survivin-K90/91R and HA-Fbxl7 for 24 h, and incubated with MG132 (20 µM) for 10 h. The WCE was subjected to survivin ubiquitination analysis. I A549 cells were transfected with Flag-Survivin-WT or Flag-Survivin-K90/91R for 24 h, followed by bergenin treated for 48 h, and incubated with MG132 (20 µM) for 10 h. The WCE was subjected to survivin ubiquitination analysis. Data were performed from at least three independent determinations and represented as mean ± sd

Akt/Wee1/CDK1 signaling pathway is required for bergenin-induced survivin ubiquitination

It has been reported that survivin phosphorylation at Thr34 residue plays an essential role in its biological function and stability. Therefore, the current study aimed to investigate whether bergenin promoted the degradation of survivin via regulating its phosphorylation. As shown in Fig. 4A, the phosphorylation of survivin was dose-dependently suppressed in NSCLC cells treated with bergenin (Fig. 4A). Moreover, it was found that the phosphorylation of Akt (Ser473 and Thr308), Wee1 (Ser642), CDK1 (Thr161; a marker for CDK1 activation) and survivin (Thr34) was attenuated following cells treatment with bergenin, while CDK1 phosphorylation at Tyr15 residue was markedly enhanced (Fig. 4B). The above inhibitory effect of bergenin was also observed in Akt-depleted NSCLC cells, (Fig. 4C), suggesting that bergenin-mediated p-Akt downregulation was responsible for the aforementioned changes in protein phosphorylation. Subsequently, to verify the above hypothesis, cells were transfected with Myr-Akt1, a constitutively activated Akt. The western blot results demonstrated that the changes in bergenin-induced phosphorylation of Wee1 (Ser642), CDK1 (Thr161 and Thr15) and survivin (Thr34) were neutralized by the exogenous activation of Akt (Fig. 4D). Additionally, Myr-Akt1 overexpression abrogated the cytotoxic effect of bergenin on NSCLC cells. The exogenous activation of Akt significantly rescued cell viability (Fig. 4E), increased the live cell population (Fig. 4F), inhibited caspase 3 activity (Fig. 4G) and reduced the protein expression levels of cleaved-caspase 3 and cleaved-PAPR (Fig. 4H), even in the presence of bergenin. Furthermore, compared with wild-type (WT) survivin, survivin mutated at the T34A residue (substitution of threonine 34 to alanine) as a nonphosphorylated mimic of survivin, notably increased its ubiquitination in bergenin-treated A549 cells (Fig. 4I). Importantly, reintroduction of survivin T34A mutation, but not WT survivin, significantly attenuated cell viability (Fig. 4J) and decreased the population of live cells in bergenin-treated NSCLC cells (Fig. 4K). The aforementioned results suggested that inhibition of the Akt/Wee1/CDK1 signal pathway induced by bergenin could be required for survivin dephosphorylation, which in turn promoted survivin ubiquitination and degradation.

Fig. 4.

Fig. 4

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Bergenin reduces survivin Thr34 phosphorylation. A A549 and H1299 cells were treated with bergenin for 48 h, followed by MG132 (20 µM) treated for 10 h, and the WCE was subjected to IB analysis. B A549 cells were treated with bergenin for 48 h, and the WCE was subjected to IB analysis. C A549 cells were transfected with siAkt for 48 h, the WCE was subjected to IB analysis. D–H A549 cells were transfected with constitutively activated Akt1 for 24 h, followed by bergenin (20 µM) treated for another 48 h, the WCE was subjected to IB analysis (D), cell viability was determined by MTS assay (E), the live cell population was tested by trypan blue exclusion assay (F) and caspase 3 activity was examined by Caspase-3 Assay Kit (G), the protein level of c-PARP and c-caspase was determined by IB analysis (H). ***p < 0.001. I A549 cells were transfected with Flag-Survivin (T34A), Flag-Survivin (WT), and His-Ub for 24 h and treated with bergenin for another 48 h. MG132 (20 µM) was added to the cell culture medium and maintained for 10 h. The WCE was subjected to survivin ubiquitination analysis. J and K. A549 cells expressing shSurvivin were transfected with Flag-survivin (WT) or Flag-survivin (T34A) mutant for 24 h, cell viability was examined by MTS assay (J), and the live cell population was tested by trypan blue exclusion assay (K). ***p < 0.001. Data were performed from at least three independent determinations and represented as mean ± sd

Bergenin induces apoptosis and inhibits tumor growth in vivo

The expression levels of cleaved-caspase 3 were detected in A549 and H1299 cells treated with different bergenin concentrations. The results showed that cleaved-caspase 3 expression (Fig. 5A) and its enzymatic activity (Fig. 5B) were both increased in a dose-dependent manner. Additionally, the intracellular localization of apoptosis-related molecules was determined, showing that bergenin could facilitate the release of cytochrome C from mitochondrial into the cytoplasm and promote the translocation of Bax to mitochondria (Fig. 5C). In addition, ectopic overexpression of survivin could compromised bergenin-induced apoptosis activation, as evidenced by the recovery of cell viability (Fig. 5D) and a decrease in caspase 3 activity (Fig. 5E) and its expression (Fig. 5F). Subcellular fractions were isolated to detect the protein expression levels of Bax and cytochrome C. The IB data revealed that cytochrome C in the cytoplasm and Bax in mitochondria were decreased following transfection of survivin (Fig. 5G). These findings suggested that the bergenin-induced activation of intrinsic apoptosis was partially mediated by survivin downregulation in NSCLC cells.

Fig. 5.

Fig. 5

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Bergenin promotes apoptosis and inhibits tumor growth in vivo. A and B A549 and H1299 cells were treated with bergenin for 48 h, the WCE was subjected to IB analysis (A), and caspase 3 activity was examined by Caspase-3 Assay Kit (B). *p < 0.05, ***p < 0.001. C A549 cells were treated with bergenin for 48 h, and subcellular fractions were isolated and subjected to IB analysis. D–G A549 cells were transfected with survivin for 24 h, followed by bergenin treated for another 48 h, cell viability was examined by MTS assay (D), caspase 3 activity was examined by Caspase-3 Assay Kit (E), the WCE was subjected to IB analysis (F), and subcellular fractions were isolated and subjected to IB analysis (G). ***p < 0.001. H-I. The tumor volume (H) and tumor weight (I) of A549-derived xenograft tumors (n = 5) were treated with vehicle control or bergenin (30 mg/kg). ***p < 0.001. J–K The tumor volume (J) and tumor weight (K) of H1299-derived xenograft tumors (n = 5) were treated with vehicle control or bergenin (30 mg/kg). ***p < 0.001. L and M IHC staining (left) and qualification (right) of Ki67 (L) and survivin positive cell population (M) in H1299-derived xenograft tumors (n = 5) with vehicle or bergenin treatment (30 mg/kg). Scale bar, 25 μm. ***p < 0.001. Data were performed from at least three independent determinations and represented as mean ± sd

Subsequently, xenograft mouse models were constructed using A549 and H1299 cells to verify the antitumor effect of bergenin in vivo. Our data demonstrated that treatment with bergenin significantly delayed the xenograft tumor growth. Compared with the vehicle-treatment group, the tumor volume was markedly reduced in the bergenin-treatment group (Fig. 5H), and tumor weight was decreased by approximately two-thirds in A549-derived xenograft tumors (Fig. 5I). Consistently, bergenin exhibited a similar significant antitumor activity in the H1299 xenograft tumor model (Fig. 5J and K). The IHC staining results in tumor sections were consistent with the in vitro observations that bergenin inhibited cell proliferation and downregulated survivin expression, as the population of Ki67 (Fig. 5L) and survivin positive cells (Fig. 5M) was significantly decreased in H1299-derived tumors. Overall, the above data indicated that bergenin could activate intrinsic apoptosis via downregulating survivin, and inhibit tumor development of NSCLC cells in vivo.

Survivin is associated with chemoresistance in NSCLC cells

Previous studies demonstrated that survivin is a critical triggering factor of drug resistance in cancer treatment [14, 46]. To determine whether survivin was involved in PEM resistance, the resistant A549- and H1299-PR NSCLC cell lines were established using parental A549 and H1299 cells. As shown in Fig. 6A, The half maximal inhibitory concentration of PEM in resistant cells was significantly higher compared with that in parental cells (Fig. 6A). Furthermore, PEM-resistant NSCLC cell lines A549-PR and H1299-PR exhibited a stronger cell viability (Fig. 6B) and colony formation capabilities (Fig. 6C and D) compared to the parental cells. We next detected the protein expression levels of survivin and found that its expression was upregulated in PEM-resistant NSCLC cells (Fig. 6E). After that, survivin stable knockdown A549-PR and H1299-PR cell lines were constructed (Fig. 6F). Depletion of survivin markedly restored the pharmacological effects of PEM on chemoresistant NSCLC cells, which was manifested by a significant decrease in cell viability (Fig. 6G), anchorage-independent cell proliferation ability (Fig. 6H) and plate colony formation (Fig. 6I). Moreover, the protein expression levels of γ-H2AX, a marker of DNA damage, were increased in survivin-depleted A549- and H1299-PR cells treated with PEM (Fig. 6J).

Fig. 6.

Fig. 6

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Survivin is correlated with chemoresistance in NSCLC cells. A The parental or resistant A549 (left) and H1299 cells (right) were treated with PEM for 48 h. The half maximal inhibitory concentration of PEM was detected by MTS assay. B MTS assay was used to determine the cell viability of A549/A549-PR and H1299/H1299-PR cells. C and D The colony formation ability of A549/A549-PR and H1299/H1299-PR cells were determined by soft agar assay (C) and plate colony formation assay (D). *p < 0.05. **p < 0.01. ***p < 0.001. E Survivin expression was determined by IB analysis in A549/A549-PR and H1299/H1299-PR cells. F Survivin expression was determined by IB analysis in A549-PR and H1299-PR cells. G-J. A549-PR and H1299-PR cells expressing shCtrl or shSurvivin treated with PEM (1 µM) for 48 h, cell viability was examined by MTS assay (G), the colony formation ability was determined by soft agar assay (H) and plate colony formation assay (I), and the WCE was subjected to IB analysis (J). ***p < 0.001. Data were performed from at least three independent determinations and represented as mean ± sd

Consistent with the in vitro results, the xenograft model derived from A549/A549-PR (Fig. 7A–C) and H1299/H1299-PR (Fig. 7D–F) cells indicated that PEM-resistant cells improved the efficacy of tumor development in vivo, as evidenced by the analysis of tumor volume (Fig. 7A and D), tumor weight (Fig. 7B and E) and the population of Ki67 positive cells (Fig. 7C and F). Additionally, as expected, depletion of survivin enhanced the sensitivity of A549-PR cells to PEM in vivo, suggesting that tumor volume (Fig. 7G) and Ki67 positive cells population (Fig. 7H) were significantly decreased in survivin-depleted NSCLC cells treated with PEM. Similarly, tumor growth was markedly reduced in the survivin-depleted H1299-PR xenograft mouse model after treatment with PEM compared with the shCtrl group (Fig. 7I and J). These results indicated that survivin could be associated with the resistance of NSCLC cells to PEM and depletion of survivin sensitized chemoresistant NSCLC cells to PEM treatment.

Fig. 7.

Fig. 7

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Knockdown survivin sensitizes NSCLC cells to PEM in vivo. A–C The tumor volume (A), tumor weight (B) and IHC staining for Ki67 positive cell population (C) of A549/A549-PR-derived xenograft tumors (n = 5). Scale bar, 25 μm. ***p < 0.001. D–F The tumor volume (D), tumor weight (E) and IHC staining for Ki67 positive cell population (F) of H1299/H1299-PR-derived xenograft tumors (n = 5). Scale bar, 25 μm. ***p < 0.001. G–H The tumor volume (G) and IHC staining for Ki67 positive cell population (H) of A549-PR expressing shCtrl or shSurvivin-derived xenograft tumors (n = 5) treated with vehicle control or PEM (3 mg/kg). Scale bar, 25 μm. Ns, p > 0.05. ***p < 0.001. I–J The tumor volume (I) and IHC staining for Ki67 positive cell population (J) of H1299-PR expressing shCtrl or shSurvivin-derived xenograft tumors (n = 5) treated with vehicle control or PEM (3 mg/kg). Scale bar, 25 μm. Ns, p > 0.05. ***p < 0.001

Bergenin overcomes chemoresistance in NSCLC cells

To further investigate whether bergenin and PEM could exert a synergistic effect on inhibiting tumor development. The present study showed that a combination of bergenin and PEM significantly attenuated cell viability (Fig. 8A) and induced DNA damage (Fig. 8B) in A549- and H1299-PR cells compared with that treated with bergenin or PEM alone. Additionally, cell treatment with bergenin impaired the colony formation ability of NSCLC cells, which was further reduced following co-treatment with PEM (Fig. 8C and D). Furthermore, it was observed that intrinsic apoptosis was markedly activated in chemoresistant cells following co-treatment with bergenin and PEM (Fig. 8E). Notably, this positive effect was also verified in the tumor xenograft mouse model. When tumor volume reached 100 mm3, A549-PR xenograft mice were administrated with bergenin, PEM or a combination of both. The results demonstrated that the tumor growth rate (Fig. 8F), tumor mass (Fig. 8G), tumor weight (Fig. 8H) and the population of Ki67-positive cells (Fig. 8I) were notably reduced in xenograft mice treated with bergenin and PEM. Moreover, a combination of bergenin and PEM treatment led to no significant weight loss in nude mice (Fig. 8J), and the H&E staining results suggested that compared with the control group, bergenin alone or in combination with PEM treatment showed no sign of toxicity to essential organ tissues, such as spleen, kidney, heart, liver and lung (Fig. 8K). These results suggested that bergenin could overcome PEM resistance in NSCLC cells and exhibit good tolerance in vivo.

Fig. 8.

Fig. 8

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Bergenin overcomes chemoresistance in NSCLC cells. A–E A549/A549-PR and H1299/H1299-PR cells were treated with PEM (1 µM), bergenin (40 µM) or a PEM/bergenin combination for 48 h. MTS assay was used to determine the cell viability (A), IB analysis to detect γ-H2AX expression (B), soft agar assay to assess the anchorage-independent cell growth (C), plate colony formation assay to analyze the colony formation (D), and IB analysis to detect c-caspase 3 expression (E). ***p < 0.001. F–K Xenograft tumors derived from A549-PR cells were treated with vehicle control (0.5% DMSO in Corn oil, 100 µL/every 2 days, i.p.), bergenin (30 mg/kg/ in 100 µL Corn oil every 2 days, i.p.), PEM (3 mg/kg/ in 100 µL Corn oil every 2 days, i.p.) or a bergenin (30 mg/kg/ every 2 days, i.p.) + PEM (3 mg/kg/ every 2 days, i.p.) combination when tumor volume reached 100 mm3. n = 5 mice per group. Tumor volume (F), tumor mass (G) and tumor weight (H) were recorded, and tumor sections were subjected to IHC staining of Ki67 (I), body weight (J) and H&E staining of major organs (K) of tumor-bearing mice were determined. Scale bar, 25 μm. ***p < 0.001. Data were performed from at least three independent determinations and represented as mean ± sd

Discussion

Clinically, the treatment strategy for patients with NSCLC generally depends on the classification and stage of the tumor, and the overall performance status of the patient [19]. Since the majority of patients diagnosed with advanced NSCLC fail to undergo surgical resection, while the proportion of patients eligible for a programmed cell death 1 (PD-1)/PD-ligand 1 immunotherapy or with the main targetable driver mutations (ALK and EGFR) is low, chemotherapeutic agents, such as cisplatin, paclitaxel, and PEM, still remain the standard treatment approach for the majority of patients with NSCLC [4, 54]. However, primary or acquired drug resistance remains the leading cause of poor response to chemotherapy [32]. Several underlying mechanisms have been identified to be responsible for chemoresistance, such as epigenetic modulations, acquisition of epithelial-mesenchymal transition (EMT) and cancer stem cell-like properties, activation of pro-survival signaling and the interaction with tumor microenvironments [32, 33, 39, 49]. A study demonstrated that calpain 2 could inhibit cell apoptosis and promote cell proliferation and migration via activating the EGFR and Akt signaling pathways, resulting in paclitaxel resistance of NSCLC [51]. Another study suggested that TIMP-1-mediated cisplatin resistance in NSCLC was associated with the regulation of mitochondrial metabolism via the acetylation of mitochondrial STAT3 and its interaction with CD44 [50]. Upregulation of the deubiquitinating enzyme UCHL1 also promoted PEM resistance by upregulating thymidylate synthase in NSCLC cells [9]. In the present study, we found that survivin expression was significantly upregulated in tumor tissues and was negatively associated with the prognosis of patients with NSCLC. Notably, survivin expression was further increased in PEM-resistant cells, while survivin depletion markedly inhibited tumor growth and re-sensitized NSCLC cells to PEM.

The regulation of survivin in tumor cells is complicated, and its expression is controlled at multiple levels, including transcriptional, translational, and posttranslational levels [21, 28]. Several transcription factors, such as SP1, NF-kB, GATA-1, STAT3, DEC1, and Notch, have been implicated as significant inducers of survivin transcription [37]. The most common signaling pathways, including but not limited to MAPK/ERK, JAK/STAT, and PI3K/AKT, play a critical role in regulating survivin expression under the action of different stimulators [31]. It has been reported that the function of survivin depends on the modification of its phosphorylation sites (Thr34, Thr117, Ser20). Protein kinase A-mediated Ser20 phosphorylation suppressed the antiapoptotic function of survivin, and the phosphorylation of Thr34 induced by cyclin-dependent kinase 1 (CDK1) was related to survivin stability and promoted its interaction with the mitotic spindle [11, 35, 47]. Moreover, ubiquitin modifications involved in regulating survivin expression have also been investigated, such as the E3 ligases Fbxl7 and XIAP [1, 3], and several deubiquitinates, including STAMBPL1, CSN5, USP1 and USP19 [6, 7, 48].

With the gradual study of the biological function and regulatory mechanism of survivin, the targeted inhibition of its expression could be a promising strategy in treating NSCLC. At present, exploring survivin inhibitors and their potential mechanisms is gaining increasing attention, and several therapeutic approaches have been developed that can interfere with survivin functionality and expression [16]. The tetracyclic aromatic core (LQZ-7F1) was synthesized by Cui et al., and exhibited an effective inhibition of survivin in a proteasome-dependent manner [36]. In addition, (-)-gossypol, a male contraceptive, sensitized hepatocellular carcinoma cells to epirubicin and downregulated survivin expression via inhibiting the ERK/4EBP1/survivin and AKT/4EBP1/p70S6K/survivin signaling pathways [18]. Furthermore, tolfenamic acid downregulated survivin expression and suppressed the development of pancreatic cancer via inhibiting the transcription factors SP1 [17]. Xanthohumol inhibited the tumorigenic properties of oral squamous cell carcinoma (OSCC) cells by reducing survivin phosphorylation at Thr34 and facilitating E3 ligase Fbxl7-mediated ubiquitination, thus resulting in survivin degradation [24]. In the present study, administration of bergenin significantly inhibited tumor growth by downregulating survivin expression. Mechanistic studies showed that bergenin inhibited the Akt/Wee1/CDK1 signaling pathway to reduce survivin phosphorylation at Thr34, promoting its interaction with E3 ligase Fbxl7, ultimately inducing survivin degradation. Moreover, co-treatment of NSCLC cells with bergenin and PEM exhibited a strong synergetic effect on inhibiting NSCLC cells proliferation to overcome chemoresistance. However, whether other molecules of the IAPs family or other modification sites of survivin are affected by bergenin treatment remains unclear. And the bioavailability of bergenin for intraperitoneal administration in mice, or any pharmacokinetics study in humans has not been reported. Therefore, further studies are needed to deeply explore the antitumor mechanism and bioavailability of bergenin.

Conclusions

In summary, the present study demonstrated that survivin, required for maintaining the malignant phenotype, was abundantly expressed in NSCLC tumor tissues and was further upregulated in chemoresistant NSCLC cell lines. Bergenin could suppresse Akt/Wee1/CDK1 signaling pathway to reduce the phosphorylation and expression of survivin, thus re-sensitizing resistant cells to PEM treatment. This study suggested that bergenin may act as an effective chemical agent targeting survivin and provided a possibility for using survivin inhibitors in combination with traditional chemotherapy agents to overcome chemoresistance for better therapeutic outcomes in patients with NSCLC.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 272 KB) (272.1KB, pdf)
Supplementary file2 (PDF 264 KB) (263.7KB, pdf)
Supplementary file3 (PDF 13261 KB) (12.9MB, pdf)

Authors' contributions

All authors contributed to the study’s conception and design. Material preparation, data collection and analysis were performed by [Xiaoying Li], [Qi Liang], [Li Zhou], [Gaoyan Deng], [Yeqing Xiao], [Yu Gan], [Shuangze Han], [Jinzhuang Liao], [Ruirui Wang], [Xiang Qing] and [Wei Li]. Writing, review, and/or revision of the manuscript was written by [Xiaoying Li], [Qi Liang], [Li Zhou], [Xiang Qing] and [Wei Li]. All authors read and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 81972837 and 82003203), the Natural Science Foundation of Hunan Province (Nos. 2021JJ31011 and 2021JJ41058) and the Hunan Province Natural Science Foundation Youth Fund Project, China (No. 2021JJ40943).

Data availability

The datasets used and analyzed in this study are available from the corresponding authors on request.

Declarations

Ethical approval

The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Third Xiangya Hospital of Central South University (Changsha, China). Informed consent was obtained from all patients.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Xiaoying Li, Qi Liang and Li Zhou contributed equally to this work.

Contributor Information

Xiang Qing, Email: qingxiang6@csu.edu.cn.

Wei Li, Email: weililx@csu.edu.cn.

References

  • 1.V. Arora, H.H. Cheung, S. Plenchette, O.C. Micali, P. Liston, R.G. Korneluk, Degradation of survivin by the X-linked inhibitor of apoptosis (XIAP)-XAF1 complex. J. Biol. Chem. 282(36), 26202–26209 (2007) [DOI] [PubMed] [Google Scholar]
  • 2.F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: Cancer J. Clin. 68(6), 394–424 (2018) [DOI] [PubMed] [Google Scholar]
  • 3.D. Camuzi, L.A. Buexm, Sd.Q.C. Lourenço, R. Grazziotin, S. Guaraldi, P. Valverde, D. Rapozo, J.M. Brooks, H. Mehanna, L.F. Ribeiro Pinto, S.C. Soares-Lima, Body hypomethylation is frequent in tumors from the digestive and respiratory tracts and is associated with risk-factor exposure. Int. J. Mol. Sci. 23(14), 7801 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.E. Cella, L. Zullo, S. Marconi, G. Rossi, S. Coco, C. Dellepiane, A. Alama, L. Rozeboom, E. Bennicelli, F. Parisi, G. Sacco, G. Barletta, L. Zinoli, M. Tagliamento, P. Pronzato, C. Genova, Immunotherapy-chemotherapy combinations for non-small cell lung cancer: current trends and future perspectives. Expert Opin. Biol. Ther. 22(10), 1259–1273 (2022) [DOI] [PubMed] [Google Scholar]
  • 5.M. Césaire, J. Montanari, H. Curcio, D. Lerouge, R. Gervais, P. Demontrond, J. Balosso, F. Chevalier, Radioresistance of non-small cell lung cancers and therapeutic perspectives. Cancers (Basel) 14(12), 2829 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.S. Chaithongyot, M. Naumann, Helicobacter pylori-induced reactive oxygen species direct turnover of CSN-associated STAMBPL1 and augment apoptotic cell death. Cell. Mol. Life Sci.: CMLS 79(2), 86 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.A.P. Chandrasekaran, A. Tyagi, N. Poondla, N. Sarodaya, J.K. Karapurkar, K. Kaushal, C.-H. Park, S.-H. Hong, K.-S. Kim, S. Ramakrishna, Dual role of deubiquitinating enzyme USP19 regulates mitotic progression and tumorigenesis by stabilizing survivin. Mol. Ther.: J. Am. Soc. Gene Ther. 30(11), 3414–3429 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.A.J. Cooper, L.V. Sequist, J.J. Lin, Third-generation EGFR and ALK inhibitors: mechanisms of resistance and management. Nat. Rev. Clin. Oncol. 19(8), 499–514 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.X. Ding, Y. Gu, M. Jin, X. Guo, S. Xue, C. Tan, J. Huang, W. Yang, M. Xue, Q. Zhou, W. Wang, Y. Zhang, The deubiquitinating enzyme UCHL1 promotes resistance to pemetrexed in non-small cell lung cancer by upregulating thymidylate synthase. Theranostics 10(13), 6048–6060 (2020) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.T. Dohi, E. Beltrami, N.R. Wall, J. Plescia, D.C. Altieri, Mitochondrial survivin inhibits apoptosis and promotes tumorigenesis. J. Clin. Investig. 114(8), 1117–1127 (2004) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.T. Dohi, F. Xia, D.C. Altieri, Compartmentalized phosphorylation of IAP by protein kinase A regulates cytoprotection. Mol. Cell 27(1), 17–28 (2007) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.N. Duma, R. Santana-Davila, J.R. Molina, Non-small cell lung cancer: epidemiology, screening, diagnosis, and treatment. Mayo Clin. Proc. 94(8), 1623–1640 (2019) [DOI] [PubMed] [Google Scholar]
  • 13.A. FäldtBeding, P. Larsson, K. Helou, Z. Einbeigi, T.Z. Parris, Pan-cancer analysis identifies BIRC5 as a prognostic biomarker. BMC Cancer 22(1), 322 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.R.N. Fuller, J. Kabagwira, P.A. Vallejos, A.D. Folkerts, N.R. Wall, Survivin splice variant 2β enhances pancreatic ductal adenocarcinoma resistance to gemcitabine. Onco. Targets Ther. 15, 1147–1160 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.H. Garg, P. Suri, J.C. Gupta, G.P. Talwar, S. Dubey, Survivin: a unique target for tumor therapy. Cancer Cell Int. 16, 49 (2016) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.R. George, S. Hehlgans, M. Fleischmann, C. Rödel, E. Fokas, F. Rödel, Advances in nanotechnology-based platforms for survivin-targeted drug discovery. Expert Opin. Drug Discov. 17(7), 733–754 (2022) [DOI] [PubMed] [Google Scholar]
  • 17.M. Hurtado, U.T. Sankpal, A. Kaba, S. Mahammad, J. Chhabra, D.T. Brown, R.K. Gurung, A.A. Holder, J.K. Vishwanatha, R. Basha, Novel survivin inhibitor for suppressing pancreatic cancer cells growth via downregulating Sp1 and Sp3 transcription factors. Cell. Physiol. Biochem.: Int. J. Exp. Cell. Physiol. Biochem. Pharmacol. 51(4), 1894–1907 (2018) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.W. Jiang, W. Wang, L. Sun, Y. Xiao, T. Ma, B. Li, X. Yan, Y. Wu, H. Li, J. Lian, F. He, (-)-Gossypol enhances the anticancer activity of epirubicin via downregulating survivin in hepatocellular carcinoma. Chem. Biol. Interact. 364, 110060 (2022) [DOI] [PubMed] [Google Scholar]
  • 19.G.S. Jones, D.R. Baldwin, Recent advances in the management of lung cancer. Clin. Med. (London, England) 18(Suppl 2), s41–s46 (2018) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.S. Khan, H.F. Bennit, N.R. Wall, The emerging role of exosomes in survivin secretion. Histol. Histopathol. 30(1), 43–50 (2015) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.F. Li, I. Aljahdali, X. Ling, Cancer therapeutics using survivin BIRC5 as a target: what can we do after over two decades of study? J. Exp. Clin. Cancer Res. 38(1), 368 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.F. Li, J. Yang, N. Ramnath, M.M. Javle, D. Tan, Nuclear or cytoplasmic expression of survivin: what is the significance? Int. J. Cancer 114(4), 509–512 (2005) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.M. Li, F. Gao, X. Li, Y. Gan, S. Han, X. Yu, H. Liu, W. Li, Correction to: stabilization of MCL-1 by E3 ligase TRAF4 confers radioresistance. Cell Death Dis. 14(1), 36 (2023) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.M. Li, F. Gao, X. Yu, Q. Zhao, L. Zhou, W. Liu, W. Li, Promotion of ubiquitination-dependent survivin destruction contributes to xanthohumol-mediated tumor suppression and overcomes radioresistance in human oral squamous cell carcinoma. J. Exp. Clin. Canc. Res. 39(1), 88 (2020) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.M. Li, H. Liu, Q. Zhao, S. Han, L. Zhou, W. Liu, W. Li, F. Gao, Targeting Aurora B kinase with Tanshinone IIA suppresses tumor growth and overcomes radioresistance. Cell Death Dis. 12(2), 152 (2021) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.W. Li, X. Yu, Z. Xia, X. Yu, L. Xie, X. Ma, H. Zhou, L. Liu, J. Wang, Y. Yang, H. Liu, Repression of Noxa by Bmi1 contributes to deguelin-induced apoptosis in non-small cell lung cancer cells. J. Cell Mol. Med. 22(12), 6213–6227 (2018) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.X. Li, Y. Wang, J. Liang, Z. Bi, H. Ruan, Y. Cui, L. Ma, Y. Wei, B. Zhou, L. Zhang, H. Zhou, C. Yang, Bergenin attenuates bleomycin-induced pulmonary fibrosis in mice via inhibiting TGF-β1 signaling pathway. Phytother. Res. 35(10), 5808–5822 (2021) [DOI] [PubMed] [Google Scholar]
  • 28.Y. Li, L.-R. He, Y. Gao, N.-N. Zhou, Y. Liu, X.-K. Zhou, J.-F. Liu, X.-Y. Guan, N.-F. Ma, D. Xie, CHD1L contributes to cisplatin resistance by upregulating the ABCB1-NF-κB axis in human non-small-cell lung cancer. Cell Death Dis. 10(2), 99 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.R. Madaan, R.K. Singla, S. Kumar, A.K. Dubey, D. Kumar, P. Sharma, R. Bala, S. Singla, B. Shen, Bergenin - A biologically active scaffold: nanotechnological perspectives. Curr. Top. Med. Chem. 22(2), 132–149 (2022) [DOI] [PubMed] [Google Scholar]
  • 30.H. Mamdani, S. Matosevic, A.B. Khalid, G. Durm, S.I. Jalal, Immunotherapy in lung cancer: current landscape and future directions. Front. Immunol. 13, 823618 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.D. Martínez-García, N. Manero-Rupérez, R. Quesada, L. Korrodi-Gregório, V. Soto-Cerrato, Therapeutic strategies involving survivin inhibition in cancer. Med. Res. Rev. 39(3), 887–909 (2019) [DOI] [PubMed] [Google Scholar]
  • 32.H.-Y. Min, H.-Y. Lee, Mechanisms of resistance to chemotherapy in non-small cell lung cancer. Arch. Pharmacal. Res. 44(2), 146–164 (2021) [DOI] [PubMed] [Google Scholar]
  • 33.H.-Y. Min, H.J. Lee, Y.-A. Suh, H. Pei, H. Kwon, H.-J. Jang, H.J. Yun, H.-G. Moon, H.-Y. Lee, Targeting epidermal growth factor receptor in paclitaxel-resistant human breast and lung cancer cells with upregulated glucose-6-phosphate dehydrogenase. Br. J. Cancer 127(4), 661–674 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.X.W. Nan, L.H. Gong, X. Chen, H.H. Zhou, P.P. Ye, Y. Yang, Z.H. Xing, M.N. Wei, Y. Li, S.T. Wang, K. Liu, Z. Shi, X.J. Yan, Survivin promotes piperlongumine resistance in ovarian cancer. Front. Oncol. 9, 1345 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.D.S. O’Connor, D. Grossman, J. Plescia, F. Li, H. Zhang, A. Villa, S. Tognin, P.C. Marchisio, D.C. Altieri, Regulation of apoptosis at cell division by p34cdc2 phosphorylation of survivin. Proc. Natl. Acad. Sci. U.S.A. 97(24), 13103–13107 (2000) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.R. Peery, Q. Cui, K. Kyei-Baffour, S. Josephraj, C. Huang, Z. Dong, M. Dai, J.-T. Zhang, J.-Y. Liu, A novel survivin dimerization inhibitor without a labile hydrazone linker induces spontaneous apoptosis and synergizes with docetaxel in prostate cancer cells. Bioorg. Med. Chem. 65, 116761 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.A. Rafatmanesh, M. Behjati, N. Mobasseri, M. Sarvizadeh, T. Mazoochi, M. Karimian, The survivin molecule as a double-edged sword in cellular physiologic and pathologic conditions and its role as a potential biomarker and therapeutic target in cancer. J. Cell. Physiol. 235(2), 725–744 (2020) [DOI] [PubMed] [Google Scholar]
  • 38.S.A. Salama, M.M. Elshafey, Cross-talk between PPARγ, NF-κB, and p38 MAPK signaling mediates the ameliorating effects of bergenin against the iron overload-induced hepatotoxicity. Chem. Biol. Interact. 368, 110207 (2022) [DOI] [PubMed] [Google Scholar]
  • 39.L. Shi, W. Zhu, Y. Huang, L. Zhuo, S. Wang, S. Chen, B. Zhang, B. Ke, Cancer-associated fibroblast-derived exosomal microRNA-20a suppresses the PTEN/PI3K-AKT pathway to promote the progression and chemoresistance of non-small cell lung cancer. Clin. Transl. Med. 12(7), e989 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.R.K. Singla, K. Dhonchak, R.K. Sodhi, M. ArockiaBabu, J. Madan, R. Madaan, S. Kumar, R. Sharma, B. Shen, Bergenin ameliorates cognitive deficits and neuropathological alterations in sodium azide-induced experimental dementia. Front. Pharmacol. 13, 994018 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Q. Sun, X. Wei, Z. Wang, Y. Zhu, W. Zhao, Y. Dong, Primary and acquired resistance against immune check inhibitors in non-small cell lung cancer. Cancers (Basel) 14(14), 3294 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 42.M. Šutić, A. Vukić, J. Baranašić, A. Försti, F. Džubur, M. Samaržija, M. Jakopović, L. Brčić, J. Knežević, Diagnostic, predictive, and prognostic biomarkers in Non-Small Cell Lung Cancer (NSCLC) management. J. Pers. Med. 11(11), 1102 (2021) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.S. Tan, Q. Gou, W. Pu, C. Guo, Y. Yang, K. Wu, Y. Liu, L. Liu, Y.-Q. Wei, Y. Peng, Circular RNA F-circEA produced from EML4-ALK fusion gene as a novel liquid biopsy biomarker for non-small cell lung cancer. Cell Res. 28(6), 693–695 (2018) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.W.D. Travis, E. Brambilla, A.G. Nicholson, Y. Yatabe, J.H.M. Austin, M.B. Beasley, L.R. Chirieac, S. Dacic, E. Duhig, D.B. Flieder, K. Geisinger, F.R. Hirsch, Y. Ishikawa, K.M. Kerr, M. Noguchi, G. Pelosi, C.A. Powell, M.S. Tsao, I. Wistuba, The 2015 World Health Organization Classification of Lung Tumors: Impact of Genetic, Clinical and Radiologic Advances Since the 2004 Classification. J. Thorac. Oncol.: Off. Publ. Int. Assoc. Study Lung Cancer 10(9), 1243–1260 (2015) [DOI] [PubMed] [Google Scholar]
  • 45.Y. Wang, X. Li, H. Wang, G. Zhang, CircCAMSAP1 promotes non-small cell lung cancer proliferation and inhibits cell apoptosis by sponging miR-1182 and regulating BIRC5. Bioengineered 13(2), 2428–2439 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.N.M. Warrier, R.K. Krishnan, V. Prabhu, R.C. Hariharapura, P. Agarwal, P. Kumar, Survivin inhibition by piperine sensitizes glioblastoma cancer stem cells and leads to better drug response. Int. J. Mol. Sci. 23(14), 7604 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.S.P. Wheatley, A.J. Henzing, H. Dodson, W. Khaled, W.C. Earnshaw, Aurora-B phosphorylation in vitro identifies a residue of survivin that is essential for its localization and binding to inner centromere protein (INCENP) in vivo. J. Biol. Chem. 279(7), 5655–5660 (2004) [DOI] [PubMed] [Google Scholar]
  • 48.S.M. Woo, S. Kim, S.U. Seo, S. Kim, J.-W. Park, G. Kim, Y.-R. Choi, K. Hur, T.K. Kwon, Inhibition of USP1 enhances anticancer drugs-induced cancer cell death through downregulation of survivin and miR-216a-5p-mediated upregulation of DR5. Cell Death Dis. 13(9), 821 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Y. Wu, Y. Zhang, D. Wang, Y. Zhang, J. Zhang, Y. Zhang, L. Xu, T. Wang, S. Wang, Q. Zhang, F. Liu, M.Y. Zaky, Q. Li, Q. Sun, D. Guo, S. Liu, L. Zou, Q. Yang, H. Liu, USP29 enhances chemotherapy-induced stemness in non-small cell lung cancer via stabilizing Snail1 in response to oxidative stress. Cell Death Dis. 11(9), 796 (2020) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.W. Xiao, P. Ahluwalia, L. Wang, J. Howard, R. Kolhe, A.M. Rojiani, M.V. Rojiani, TIMP-1 dependent modulation of metabolic profiles impacts chemoresistance in NSCLC. Cells 11(19), 3036 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.F. Xu, J. Gu, C. Lu, W. Mao, L. Wang, Q. Zhu, Z. Liu, Y. Chu, R. Liu, D. Ge, Calpain-2 enhances non-small cell lung cancer progression and chemoresistance to paclitaxel via EGFR-pAKT pathway. Int. J. Biol. Sci. 15(1), 127–137 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.S. Yang, Z. Yu, L. Wang, T. Yuan, X. Wang, X. Zhang, J. Wang, Y. Lv, G. Du, The natural product bergenin ameliorates lipopolysaccharide-induced acute lung injury by inhibiting NF-kappaB activition. J. Ethnopharmacol. 200, 147–155 (2017) [DOI] [PubMed] [Google Scholar]
  • 53.X. Yu, R. Wang, Y. Zhang, L. Zhou, W. Wang, H. Liu, W. Li, Skp2-mediated ubiquitination and mitochondrial localization of Akt drive tumor growth and chemoresistance to cisplatin. Oncogene 38(50), 7457–7472 (2019) [DOI] [PubMed] [Google Scholar]
  • 54.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, 61 (2019) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Q. Zeng, T. Zhou, F. Zhao, D. Xiong, B. He, Q. Hua, M. Lin, L. Deng, X. Sang, W. Xie, J. Chen, Z. Wang, L. Ren, Z. Luo, X. Huang, W. Liu, S. Tang, p62-Nrf2 regulatory loop mediates the anti-pulmonary fibrosis effect of bergenin. Antioxidants (Basel) 11(2), 307 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.J. Zhang, Y. Nishimoto, H. Tokuda, N. Suzuki, K. Yasukawa, W. Kitdamrongtham, H. Akazawa, A. Manosroi, J. Manosroi, T. Akihisa, Cancer chemopreventive effect of bergenin from Peltophorum pterocarpum wood. Chem. Biodivers. 10(10), 1866–1875 (2013) [DOI] [PubMed] [Google Scholar]
  • 57.Y. Zhang, Y. Sun, Y. Jia, Q. Zhang, P. Zhu, X. Ma, α5-nAChR and survivin: Two potential biological targets in lung adenocarcinoma. J. Cell. Physiol. 236(3), 1787–1797 (2021) [DOI] [PubMed] [Google Scholar]
  • 58.H. Zhou, Q. Guan, X. Hou, L. Liu, L. Zhou, W. Li, H. Liu, Epithelial-mesenchymal reprogramming by KLF4-regulated Rictor expression contributes to metastasis of non-small cell lung cancer cells. Int. J. Biol. Sci. 18(13), 4869–4883 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Y. Zhu, Z. Ouyang, H. Du, M. Wang, J. Wang, H. Sun, L. Kong, Q. Xu, H. Ma, Y. Sun, New opportunities and challenges of natural products research: When target identification meets single-cell multiomics. Acta Pharm. Sinica B 12(11), 4011–4039 (2022) [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary file1 (PDF 272 KB) (272.1KB, pdf)
Supplementary file2 (PDF 264 KB) (263.7KB, pdf)
Supplementary file3 (PDF 13261 KB) (12.9MB, pdf)

Data Availability Statement

The datasets used and analyzed in this study are available from the corresponding authors on request.

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