Wogonin derivative V8 enhances bortezomib efficacy in gastric carcinoma by disrupting lysosome-mediated drug resistance

Abstract

BACKGROUND

Bortezomib (BTZ) is ineffective in gastric carcinoma (GC) due to lysosome-mediated resistance. Wogonin derivative V8 targets lysosomes.

AIM

To address the limited efficacy of BTZ in GC and explore whether wogonin derivative V8 enhances anti-GC effects by overcoming lysosome-mediated resistance.

METHODS

In vitro experiments used four human GC cell lines (MGC-803, BGC-823, AGS, and HGC-27) to assess cell viability (cell counting kit-8), lysosomal function (LysoSensor staining), autophagy (western blotting for light chain 3/p62), and drug synergy [combination index (CI)]. Liquid chromatography/mass spectrometry measured intracellular BTZ concentration. Transcription factor EB (TFEB), a master regulator of lysosomal and autophagy genes, was investigated using small interfering RNA silencing and plasmid overexpression. In vivo efficacy/safety was tested in MGC-803 xenograft nude mice, and patient-derived tumor organoids (PDOs) validated clinical relevance.

RESULTS

BTZ was trapped in GC cell lysosomes, reducing its proteasome accessibility and inducing resistance; lysosome number correlated positively with the half-maximal inhibitory concentration of BTZ. V8 induced lysosomal damage/deacidification, increasing intracellular BTZ availability. V8 + BTZ synergistically inhibited GC cell growth (CI < 1), upregulated proteotoxic stress markers (ATF4, immunoglobulin heavy chain binding protein) and apoptotic mediators (cleaved caspase-3). TFEB knockdown enhanced V8 + BTZ cytotoxicity, whereas its overexpression reduced cell death, indicating a protective role of TFEB in gastric cancer cells. In xenografts, V8 + BTZ significantly reduced tumor volume/weight (P < 0.01) without organ toxicity. PDOs showed enhanced sensitivity to V8 + BTZ vs monotherapy.

CONCLUSION

Lysosomes mediate BTZ resistance in GC; V8 overcomes this by disrupting lysosomes, enabling BTZ to target proteasomes. V8 + BTZ is a safe, effective strategy against GC.

Key Words: Gastric carcinoma; Bortezomib; Wogonin derivative V8; Lysosome; Drug resistance; Transcription factor EB; Patient-derived organoids

Core Tip: We identified lysosomal trapping as a key mechanism of bortezomib resistance (BTZ) in gastric cancer. The novel agent V8 disrupts lysosomes, releasing BTZ to enhance proteasome targeting and induce lethal proteotoxic stress. The V8-BTZ combination shows strong synergy in cells, xenografts, and patient organoids, supporting its clinical potential for overcoming resistance.

INTRODUCTION

Gastric carcinoma is a common digestive system tumor with high incidence and mortality rates worldwide. Although early diagnosis and various treatment options depending on disease stages improve the prognosis of gastric cancer, the 5-year survival rate remains at 25%[1]. Currently, more effective treatment still needs to be developed.

Bortezomib (BTZ) (Velcade^®) is an effective and highly selective proteasome inhibitor, emerging as front-line therapy in multiple myeloma (MM). However, it has demonstrated limited efficacy in solid cancer[2-6]. This could be because cancer cells developed the ability to adapt to transient proteasome inhibition. Multiple mechanisms have been associated with cellular resistance to proteasome inhibition, such as constitutive activation of nuclear factor-κB, increased proteasome activity, and dysregulation of proteasome catalytic subunits[7-9]. In addition, mitochondrial metabolism serves as a nongenetic contributor to cellular adaption to proteasome inhibition[10]. Thus, modulating resistance-associated pathway might broaden the application of BTZ on solid cancer treatment in clinic.

As an acidic membrane-bound organelle, lysosome has been suggested to mediate drug resistance[11]. Activation of the autophagy-lysosome pathway acts as survival mechanism under various drug treatments. BTZ can induce autophagy, which contributes to reduced drug sensitivity in MM cells[12-14]. Beyond macro-autophagy, lysosomes also play a central role in initiating specific forms of regulated cell death, notably lysosomal cell death. Lysosomal cell death is characterized by lysosomal membrane permeabilization, leading to the release of cathepsins and other hydrolases into the cytosol, which triggers oxidative stress, mitochondrial dysfunction, and ultimately caspase-dependent or -independent cell death[15,16]. Transcription factor EB (TFEB) is a master regulator of autophagy-lysosome pathway, and functions through regulating gene network related with autophagy and lysosome biogenesis[17]. Not surprisingly, a recent report found that BTZ activated TFEB signaling pathway in MM cells[18]. Nevertheless, involvement of the autophagy–lysosome pathway in resistance of solid cancer cells against BTZ treatment remains to be further investigated.

Wogonin is a major bioactive compound isolated from the traditional Chinese medicinal plant Scutellaria baicalensis, and exerts an anticancer effect through regulating multiple signaling pathways[19,20]. Preclinically accumulating evidence showed that natural flavonoids and nonflavonoid polyphenols, including wogonin, can exhibit a synergistic effect on MM therapy with BTZ[21]. However, whether wogonin and its analogs increases chemosensitivity of BTZ in solid cancer remains unknown. We recently identified that wogonin derivate V8 exhibited potential anticancer effects through inducing lysosomal cell death[22]. In the current study, we found that V8-induced lysosomal damage enhanced the cytotoxicity of BTZ in gastric cancer cells because the latter is susceptible to be trapped in lysosomes, therefore reducing accessibility to proteasomes. V8 displayed synergistic action with BTZ on tumor growth in vitro and in vivo, and combination of them also suppressed the growth of patient-derived tumor organoids.

MATERIALS AND METHODS

Reagents

V8 (C₂₄H₂₉NO₇, molecular weight: 443.49, purity ≥ 99%) was synthesized as described previously[23]. Wogonin was kindly gifted from Prof. Li ZY (China Pharmaceutical University). BTZ (HY-10227), benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (Z-VAD-FMK) (HY-16658B), chloroquine (HY-17589A), 3-methyladenine (3-MA) (HY-19312), bafilomycin A1 (Baf-A1) (HY-100558) and L-leucyl-L-leucine methyl ester hydrochloride (HY-129905) were purchased from MedChemExpress (Shanghai, China). Cell counting kit-8 (CCK-8) solution was obtained from Vazyme (A311-01/02, Nanjing, China). Primary antibodies against ATF4, Bax, immunoglobulin heavy chain binding protein (Bip), Bcl-xL, β-actin, caspase-3, cleaved-caspase-3, caspase-9, caspase-12, cleaved-caspase-12, phosphorylated eukaryotic translation initiation factor 2 alpha (p-eIF2α), p62, TFEB, microtubule-associated protein 1A/1B-light chain 3 (LC3), Ki67 and horseradish-peroxidase-conjugated goat anti-mouse/rabbit secondary antibodies were obtained from Abclonal Technology (Wuhan, Hubei Province, China); primary antibodies against caseinolytic mitochondrial matrix peptidase proteolytic subunit (Clpp), LAMP1, and ubiquitin were from Proteintech (Wuhan, Hubei Province, China).

Cell culture

The human MGC-803, BGC-823 cell lines were purchased from the Shanghai Institute of Cell Biology and Chinese Academy of Sciences (Shanghai, China). The human AGS and HGC-27 cell lines were purchased from Cellcook Biotech. Co. Ltd. (Guangzhou, Guangdong Province, China). All cells were authenticated using short tandem repeat or single nucleotide polymorphism profiling. MGC-803 and BGC-823 cells were cultured in Roswell Park Memorial Institute-1640 medium (31800022; Thermo Fisher Scientific, Gibco). AGS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) F-12 medium (11320033; Thermo Fisher Scientific, Gibco). HGC-27 cells were cultured in DMEM (12100061; Thermo Fisher Scientific). All media were supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin and 100 μg/mL streptomycin (Solarbio Science and Technology Co. Ltd., Beijing, China). Cells were cultured in a humidified environment with 5% carbon dioxide (CO₂) at 37 °C, and routinely tested for mycoplasma.

Cell proliferation assay

MGC-803, BGC-823, AGS, and HGC-27 cells were seeded into 96-well plates at a density of 5000 cells/well. The cells were treated with various concentration of V8 and BTZ for 24 hours. After treatment, 10 μL CCK-8 solution was added to 100 μL culture medium in each well, and plates were maintained at 37 °C for a further 1 hour. The absorbance (A) at 450 nm was measured by a microplate reader (BioTek Instruments, Winooski, VT, United States). The inhibition ratio was calculated as [(A_control - A_treated)/A_control] × 100%. The combination index was calculated according to the method of Chou[24], using the Calcusyn software (Biosoft, United Kingdom).

Liquid chromatography and mass spectrometry

MGC-803 cells were treated with BTZ and Baf-A1 alone, or in combination for 5 hours. The harvested cells were lysed by repeated freeze-thaw cycles, followed by mixing with internal standard solution. BTZ was extracted by prechilled acetonitrile (containing 0.1% formic acid) for 10 minutes on ice. After centrifugation at 12000 × g for 10 minutes at 4 °C, the sample was diluted following nitrogen drying of supernatant and analyzed by liquid chromatography (LC-20A; Shimadzu, Japan) coupled with mass spectrometer (LTQ-XL; Thermo Fisher). The ultimate AQ-C18 chromatographic column (3 μm, 2.1 mm × 150 mm) (YueXu Technology, Shanghai) was applied for separation under the following conditions: Elution (solvent A = 0.1% formic acid in water and solvent B = 0.1% formic acid in acetonitrile), injection volume of 10 μL, flow rate of 300 μL/minute, and temperature 40 °C. The source conditions for positive ion electrospray ionization mode selected reaction monitoring were optimized as follows: Ion source temperature 275 °C, sheath gas 24 arb, auxiliary gas 0 arb, first mass scan range 50-1000 Da, and second mass fragmentation energy 35 eV. Standard solution of BTZ (10 μg/mL) was prepared, and the peak area of BTZ (m/z 367 to 226) in selected ion monitoring mode was 837065, and the absolute intensity of the internal standard (m/z 235 to 86) had a peak area of 608592, with a relative ratio of 1.3754. The intracellular concentration of BTZ was calculated by a formula: C_sample = (R_sample/1.3754) × C_standard × dilution factor, where R_sample represents the ratio of the peak area of BTZ in samples to that of internal standard, C_standard equals to 10 μg/mL.

Flow cytometry

Cells treated with V8 and BTZ alone or combination were harvested and stained with the Annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit (A211-01/02; Vazyme, Nanjing, Jiangsu Province, China), LysoSensor^TM Green DND-189 (40767ES50; Yeasen Biotechnology). After that, cells were subjected to fluorescence-activated cell sorting Calibur flow cytometry (BD Bioscience, Franklin Lakes, NJ, United States) to analyze cell death (Annexin V⁺ and/or PI⁺ cells) and lysosomal acidity and the data analysis was performed by FlowJo software (Treestar, Ashland, OR, United States).

Western blotting

Cells were lysed using radio immunoprecipitation assay buffer (89901; Thermo Fisher Scientific) supplemented with proteinase inhibitor cocktails (GK10014; GlpBio, Montclair, CA, United States). After determining protein concentration using bicinchoninic acid methods, an equal amount of protein was mixed with loading buffer (3:1, v/v) and boiled for 5 minutes. The denatured samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were cut horizontally according to target protein size. After blocking with 3% bovine serum albumin (BSA) for 1 hour at room temperature, the membranes were incubated with primary antibodies overnight at 4 °C, followed by three washes using phosphate-buffered saline (PBS)-tween (PBST) solution. Secondary antibody was added for 1 hour at room temperature. After washing with PBST, the protein bands were visualized using enhanced chemiluminescence solution (E411-04/05; Vazyme) and photographed by Amersham Imager 600 (GE Healthcare, Buckinghamshire, United Kingdom).

Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 30 minutes and washed with PBS containing 0.3% triton X-100 three times (10 minutes each). After blocking with 5% BSA for 1 hour at room temperature, the cells were incubated with primary antibody overnight at 4 °C. After washing, the samples were incubated with Alexa fluor secondary antibody for 1 hour and counter-stained with 4’,6-diamidino-2-phenylindole. The samples were imaged using confocal laser scanning microscope (FV1000; Olympus, Tokyo, Japan).

Plasmid transfection, RNA interference and lentivirus system

The small interfering RNA (siRNA) pairs against TFEB and CTSB genes were purchased from GenePharma (Shanghai, China). The sequences are listed as follows: SiCtrl-sense: 5’-UUCUCCGAACGUGUCAACGU-3’; siTFEB-sense: 5’-AGACGAAGGUUCAACAUCA-3’; siCTSB-sense: 5’-GGCACAACUUCUACAACGUTT-3’. The plasmid expressing TFEB (p-enhancer-cytomegalovirus (pEnCMV)-TFEB-wild-type and empty vector (pEnCMV-empty) were obtained from MiaoLing Plasmid Platform (Wuhan, Hubei Province, China). Constitutively active form of TFEB (pEnCMV-TFEB-S211A) was made by ClonExpress II kit (Vazyme) using the following primers: P1-forward: 5’-TCCCAATGCCCCCATGGCCATGCTGCACATTGGCT-3’; P1-reverse: 5’- TGGCCATGGGGGCATTGGGAGCACTATTGCCAGCG-3’; P2-forward: 5’- GCCCAACCTGCCATCACGAGATTTCGAT-3’; P2-reverse: 5’- ATCGAAATCTCGTGATGGCAGGTTGGGC-3’. All constructs were confirmed by sequencing (GENEWIZ, Suzhou, Jiangsu Province, China). The plasmid (2 μg) or siRNA (400 pmol) per well in a six-well plate was transfected into the cells with lipofectamine 2000 (11668019; Invitrogen, Carlsbad, CA, United States). The lentivirus containing mCherry-green fluorescent protein (GFP)-LC3B was purchased from Hanbio Biotechnology Co. Ltd. (Shanghai, China).

Animal study

Female athymic BALB/c nude mice (4-6 weeks old) weighing 18-22 g were purchased from CAVENS Laboratory Animals (Changzhou, Jiangsu Province, China). Animals were raised under asterile environment (21-25 °C with 50%-60% humidity) on a 12 hours light/dark cycle with free access to food and water. All animal experiments were approved by the Animal Ethics Committee of China Pharmaceutical University (No. 2024-08-093). MGC-803 cells (2.5 × 10⁶) were subcutaneously injected into the right flank of mice to establish the xenograft model. The mice were randomly divided into four groups (5 mice per group). The control group was treated with physiological saline, and V8 (75 mg/kg) was given by gastric gavage every day, while BTZ (0.75 mg/kg)[25-27] was intraperitoneally injected every 3 days. The tumor volume was recorded every 3 days, which was calculated by the formula: (L × W²)/2, where L was the largest diameter and W the diameter perpendicular to L. After treatment for 3 weeks, mice were killed and tumor, heart, liver, kidney and blood samples were harvested for analysis.

Immunohistochemical analysis

Tissues were fixed with 4% paraformaldehyde solution, embedded in paraffin, and cut into 4-μm sections. Hematoxylin and eosin (HE) staining was used to estimate tissue morphological characteristics. For detecting protein expression, slides were deparaffinized and rehydrated, and treated in a microwave in distilled water for 2 minutes before blocking by 10% goat serum for 30 minutes at room temperature. The sections were subsequently incubated with primary antibodies against Ki67 (A2092, 1:300; Abclonal), LAMP1 (553792, 1:200; Proteintech), and cleaved caspase-3 (Abclonal, AC033, 1:250) at 4 °C overnight, followed by visualization using an immunohistochemistry kit (PK10006; Proteintech). All sections were photographed under an Axio Scope.A1 microscope (Zeiss, Oberkochen, Germany). Immunohistochemical quantification: Using ImageJ software, five different fields per section were selected to calculate the ratio of Ki67-positive cells to total nuclei. Average optical density was expressed as the ratio of positively stained area to nuclear-stained area.

Patient-derived tumor organoids

Gastric cancer tissue samples were obtained from Nanjing Jiangning Hospital (Nanjing, Jiangsu Province, China) with informed consent from patients. The study was approved by the Ethics Committee of Nanjing Jiangning Hospital (No. 2024-03-150-K01). Patient tissues were washed with cold DMEM/F12 (Gibco), fragmented using a scalpel or razor blades into 1-2 mm³ pieces and digested at 37 °C for 30 minutes with solution containing type II and IV collagenase (0.5 mg/mL, Worthington), DNase I (0.1 mg/mL, Worthington), and penicillin-streptomycin (100 U/mL, Life Technologies). After filtering the suspension through a 100-μm strainer, the collected cells were resuspended in Matrigel (Corning) and cultured using organoid medium in a humidified incubator with 5% CO₂ at 37 °C. The organoids were cultured using advanced DMEM/F12 (Gibco) media supplemented with 10 mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Gibco), 1 × GlutaMAX (Gibco), 100 U/mL penicillin-streptomycin (Gibco), 1 × B27 (Gibco), 50 ng/mL epidermal growth factor (Novoprotein), 100 ng/mL noggin (Novoprotein), 500 nM A83-01 (Beyotime), 100 ng/mL Wnt3a (Novoprotein), 500 ng/mL R-spondin 1 (Novoprotein), 10 nM gastrin I (TOCRIS), 1.25 mmol/L N-acetylcysteine (Sigma), 10 mmol/L nicotinamide (Sigma), and 10 μM Y-27632 (Beyotime). The medium was changed every 3 days. For passaging, organoids were digested with TrypLE Express (Gibco) and split every 7-10 days at a ratio of 1:3 or 1:4. For testing drug sensitivity, organoids were partially digested and collected. The cell clusters were resuspended in the organoid culture medium with 10% matrigel, and 50-100 clusters were seeded per well of 384-well plates, and cultured for 2 days before drug treatment. Each treatment was in triplicate. Images were taken by IX73 microscope (Olympus). Cell viability was examined using cell counting-lite three-dimensional luminescent cell viability assay (Vazyme).

Statistical analysis

All data were presented as mean ± SD. Statistical analysis was performed with GraphPad Prism 8.0 software. For comparisons of three or more groups, one-way analysis of variance was performed with Tukey’s multiple comparisons test. For comparisons of two groups, a two-tailed unpaired t test was performed. Significance compared with control group was described as ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001 and no significance.

RESULTS

Effect of autophagy inhibitors on BTZ-induced cell death

Previous reports have shown that most gastric cancer cells are resistant to BTZ (Figure 1A) treatment in vitro[28,29]. We showed that BTZ at a high concentration (100 nM) only induced around 20% MGC-803 cell death (Figure 1B and C), and no significant change in cell death was recorded with higher concentrations, suggesting activation of a pro-survival pathway. We determined whether autophagy could be involved in resistance of MGC-803 cells to BTZ. BTZ upregulated autophagy marker p62 and LC3-II expression (Figure 1D). Formation of mCherry-GFP-LC3 puncta as an indicator of autophagosomes was also increased by BTZ treatment (Figure 1E). However, inhibition of autophagosome formation by 3-MA only slightly altered BTZ induced cell death (Figure 1F and G). By contrast, chloroquine, a well-known inducer of lysosomal damage, caused a dramatic increase (about twofold) in cell death in the presence of BTZ (Figure 1H and I). Similar results were observed when combining BTZ with vacuolar hydrogen ion-translocating adenosine triphosphatase inhibitors Baf-A1 and ammonium chloride; both of which were able to deacidify lysosome, triggering its dysfunction (Supplementary Figure 1). These results suggest that the reduced sensitivity of gastric cancer cells to BTZ is more closely associated with lysosome dysfunction than with a protective autophagy response.

Figure 1 Effect of bortezomib on apoptosis and autophagy in MGC-803 gastric cancer cells. A: Structure of bortezomib (BTZ); B and C: MGC-803 cells were treated with different concentration of BTZ for 24 hours, followed by flow cytometry for cell death. Representative images (B) and quantification data (C) are shown; D: MGC-803 cells were treated with BTZ (100 nM). The cell lysates were subjected to western blotting using antibody against p62 and light chain 3 (LC3), and β-actin as loading control; E: MGC-803 cells were transfected using lentivirus containing mCherry- green fluorescent protein-LC3, and treated with BTZ (100 nM) for 24 hours. Immunofluorescence analysis was performed for evaluating autophagosome (scale bar: 20 μm); F-I: MGC-803 cells were treated with BTZ (100 nM) with or without 3-methyladenine (5 mmol/L), and chloroquine (40 μM) for 24 hours, followed by cell death detection. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. BTZ: Bortezomib; DMSO: Dimethyl sulfoxide; V-FITC: Annexin V-fluorescein isothiocyanate; PI: Propidium iodide; LC3: Light chain 3; NS: No significance; GFP: Green fluorescent protein; 3-MA: 3-methyladenine; CQ: Chloroquine.

Lysosome mediates resistance of gastric cancer cells to BTZ

The weakly basic property of BTZ (pKa = 8.64) means that it might be trapped in the lysosomal lumen, resulting in reduced accessibility of proteasomes. As expected, the intracellular concentration of BTZ dramatically dropped due to the presence of Baf-A1, implying that BTZ can be sequestrated into lysosomes (Figure 2A). Nevertheless, BTZ did not cause lysosomal stress as indicated by staining with Lyso-Sensor Green dye, but seemed to increase the number of lysosomes (Figure 2B and C). A previous study showed that some chemotherapeutic agents at nanomolar concentrations promoted TFEB-dependent lysosomal biogenesis[30], which agrees with our observation on the upregulation of TFEB downstream gene expression (Supplementary Figure 2A). However, silencing TFEB expression did not promote BTZ-induced apoptosis (Supplementary Figure 2B-D). It is likely that existing lysosomes are sufficient to tolerate BTZ treatment. To further study the contribution of lysosomes to BTZ resistance, we analyzed the number of lysosomes through staining of LAMP1; a marker lysosomal transmembrane protein in different human gastric cancer cell lines (Figure 2D). The number of lysosomes varied in different cell lines (Figure 2E). BTZ consistently suppressed cell proliferation in all four cell lines (Figure 2F). Spearman’s correlation analysis found that lysosome number had a significantly positive link with the half-maximal inhibitory concentration of BTZ (Figure 2G). Our results suggested that lysosome mediated BTZ resistance in gastric cancer cells.

Figure 2 Lysosome affects the anticancer effect of bortezomib. A: Intracellular drug concentration was examined using liquid chromatography/mass spectrometry after cells were treated with bortezomib (BTZ) (100 nM) and bafilomycin A1 (100 nM) alone or in combination for 6 hours; B and C: MGC-803 cells were treated with indicated concentration of BTZ and L-leucyl-L-leucine methyl ester hydrochloride (1 mmol/L) for 6 hours, followed by staining of LysoSensor Green DND-189. Representative images (B) and quantification data (C) of flow cytometry were shown; D and E: Representative images (D) and quantified results (E) of immunofluorescent analysis for LAMP1 expression in different gastric cancer cell lines (scale bar: 10 μm); F: Cell viability assay was performed in different gastric cancer cells treated with BTZ; G: Spearman correlation was determined between half-maximal inhibitory concentration value for BTZ and cellular LAMP1 expression. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. BTZ: Bortezomib; Baf-A1: Bafilomycin A1; LLOMe: L-leucyl-L-leucine methyl ester hydrochloride; DMSO: Dimethyl sulfoxide; MFI: Mean fluorescence intensity; DAPI: 4’,6-Diamidino-2-phenylindole; NS: No significance; IC₅₀: Half-maximal inhibitory concentration.

V8 induces lysosomal cell death in gastric cancer cells

We investigated whether a synthetic flavonoid derivate V8 with lysosomotrophic property (Figure 3A) induced lysosome damage, therefore promoting chemosensitivity to BTZ in gastric cancer cells. There was increased cell death in the different gastric cancer cells in response to V8 treatment (Figure 3B and C). In contrast to BTZ, V8 led to lysosomal alkalinization, similar to traditional lysosome inhibitors (Figure 3D and E). Lysosomal damage was aggravated by V8 treatment in MGC-803 cells, as indicated by staining of Lyso-Tracker Red dye (Figure 3F and G). These events were associated with cellular vacuolation (Supplementary Figure 3A), the most notable feature of most lysosomotropic molecules, and autophagic blockage at a late stage in the presence of V8 (Supplementary Figure 3B). Cathepsin B, a major lysosomal protease, was released into the cytoplasm to trigger cell death upon lysosome membrane permeability[31]. As expected, silencing CSTB gene expression in MGC-803 cells partially ameliorated V8-induced apoptosis (Figure 3H and I; Supplementary Figure 3C). Pretreatment with Baf-A1, which suppressed V8 accumulation in lysosomes, almost completely inhibited the prodeath effect of V8 in different gastric cancer cells (Figure 3J and K; Supplementary Figure 3D-I). These results demonstrated that V8 treatment resulted in death of gastric cancer cells through induction of lysosomal destruction.

Figure 3 V8 induces lysosomal cell death in gastric cancer cells. A: Structure of synthetic flavonoid derivate V8; B and C: Gastric cancer cells were treated with V8 at indicated concentration for 24 hours. Representative images (B) and quantified data (C) for cell death are shown; D and E: MGC-803 cells were treated with V8 and L-leucyl-L-leucine methyl ester hydrochloride (1 mmol/L) for 6 hours, followed by staining with LysoSensor Green DND-189 dye. Representative images (D) and quantification data (E) are shown; F and G: MGC-803 cells were treated with V8 (9 μM), followed by staining with LysoTracker red dye. Representative images and quantified intensity of immunofluorescence analysis are shown (scale bar: 25 μm); H-K: MGC-803 cells were transfected with small interfering RNA against CSTB gene or treated with bafilomycin A1 (100 nM), followed by V8 (12 μM) for 24 hours. Representative images (H and J) and quantification data (I and K) of flow cytometry analysis for cell death are shown. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. ^dP < 0.0001. V-FITC: Annexin V-fluorescein isothiocyanate; PI: Propidium iodide; LLOMe: L-leucyl-L-leucine methyl ester hydrochloride; DMSO: Dimethyl sulfoxide; MFI: Mean fluorescence intensity; DAPI: 4’,6-Diamidino-2-phenylindole; NS: No significance; Baf-A1: Bafilomycin A1; siCtrl: Negative control small interfering RNA; siCTSB: Small interfering RNA targeting CTSB.

V8 and BTZ acts synergistically on gastric cancer cell in vitro

To explore whether V8 and BTZ had synergistic action on gastric cancer cells, we evaluated the cell viability of four gastric cancer cell lines treated with different concentration of V8 and BTZ. Combination of V8 and BTZ caused apparent death in all cells (Figure 4A). The synergistic effect of V8 combined with BTZ was evaluated by calculating combination index. V8 and BTZ synergistically affected gastric cells with a high number of lysosomes (Figure 4B). Wogonin, a parent compound of V8, had little impact on BTZ induced cell death, similar to V8 at the same concentration, which could be attributed to less lysosomal sequestration (Supplementary Figure 4A-D). Lysosomal damage led to BTZ release, thereby improving its accessibility to proteasomes. Expectedly, the molecular markers for protein quality control in endoplasmic reticulum (ER) (ATF4, Bip, p-eIF2α) and mitochondria (Clpp), and total ubiquitin-modified proteins were upregulated by combination treatment with V8 and BTZ, suggesting that the level of cellular misfolded proteins was increased (Figure 4C). Consequently, activation of apoptosis mediators associated with proteotoxic stress was enhanced by combination treatment with V8 and BTZ (Figure 4D). Reactive oxygen species (ROS) seemed not to be involved in cell death induced by combined V8 and BTZ (Supplementary Figure 4E and F). Inhibition of caspase activity using Z-VAD-FMK (pan-inhibitor of caspase) markedly reduced cell death induced by V8 and BTZ combination (Figure 4E). Combined V8 and BTZ treatment induced integrated cellular stress encompassing ER stress, mitochondrial dysregulation, and ubiquitinated protein accumulation reflecting severe proteotoxic stress (Figure 4C). While ROS contributed minimally, caspase-dependent apoptosis was central to cell death, as indicated by Z-VAD-FMK-mediated rescue (Figure 4F). These results demonstrate that V8 and BTZ act synergistically to disrupt proteostasis and trigger apoptosis, and improve anticancer effect on gastric cancer cells; probably attributed to lysosomal dysfunction.

Figure 4 V8 synergizes with bortezomib to inhibit gastric cancer cell growth. A: Cell viability assay was performed in different gastric cancer cells treated with V8 and bortezomib (BTZ) for 24 hours; B: Combination index was calculated in different gastric cancer cells; C and D: MGC-803 cells were treated with V8 (9 μM) and BTZ (100 nM) for 24 hours. Western blotting was carried out for analysis of indicated protein expression with β-actin as loading control; E and F: Cells were treated with V8 (9 μM), BTZ (100 nM), or benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone (20 μM) for 24 hours, followed by flow cytometry assay for apoptosis analysis. Representative images (E) and quantitative data (F) are shown. ^bP < 0.01. ^dP < 0.0001. CI: Combination index; Bip: Immunoglobulin heavy chain binding protein; p-eIF2α: Phosphorylated eukaryotic translation initiation factor 2 alpha; Clpp: Caseinolytic mitochondrial matrix peptidase proteolytic subunit; DMSO: Dimethyl sulfoxide; BTZ: Bortezomib; V-FITC: Annexin V-fluorescein isothiocyanate; PI: Propidium iodide; Z-VAD-FMK: Benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone.

TFEB modulates the combined effect of V8 and BTZ on gastric cancer cells

Given the central roles of TFEB in lysosomal stress response, we investigated its influence on the cytotoxicity of combined V8 and BTZ. TFEB is usually phosphorylated at S211 by mammalian target of rapamycin complex 1 and retained in the cytosol by binding to 14-3-3 protein[32]. Under stressful conditions, TFEB is translocated to the nucleus, where it increases expression of autophagy and lysosomal biogenesis-related genes by binding to coordinated lysosomal expression and regulation element[17]. TFEB-S211A mutant form, which has disrupted phosphorylation, showed constitutive nuclear localization (Figure 5A). Overexpression of TFEB-S211A significantly reduced apoptosis by V8 or BTZ alone in MGC-803 cells compared with that of wild-type TFEB; probably because of enhanced lysosomal biogenesis (Supplementary Figure 5). Cell apoptosis was attenuated by ectopic expression of TFEB-S211A in the presence of combined V8 and BTZ (Figure 5B-D). Silencing endogenous TFEB expression to block lysosomal biogenesis significantly augmented apoptosis induced by combination of V8 and BTZ (Figure 5E-G). These results demonstrated that alteration of the TFEB signaling pathway modulated the cytotoxicity of combined V8 and BTZ; probably through affecting lysosomal biogenesis.

Figure 5 Role of transcription factor EB in cell apoptosis induced by V8 and bortezomib. A: MGC-803 cells were transiently transfected with empty vector, wild-type, and S211A mutant form of transcription factor EB (TFEB) plasmids. Representative images of immunofluorescence assay are shown for analysis of cellular TFEB localization (scale bar: 30 μm); B-D: MGC-803 cells were transiently transfected with indicated plasmids (B), followed by treatment of V8 (9 μM) together with bortezomib (BTZ) (100 nM) for 24 hours. Representative images (C) and quantitative results (D) are shown; E: TFEB expression was examined by western blotting after transfection of specific small interfering RNA in MGC-803 cells; F and G: MGC-803 cells were treated by V8 (9 μM) combined with BTZ (100 nM) for 24 hours. Representative images (F) and quantified results (G) of cell apoptosis analysis are shown. ^bP < 0.01. DAPI: 4’,6-Diamidino-2-phenylindole; TFEB: Transcription factor EB; EV: Empty vector; WT: Wild-type; DMSO: Dimethyl sulfoxide; BTZ: Bortezomib; V-FITC: Annexin V-fluorescein isothiocyanate; PI: Propidium iodide; siCtrl: Negative control small interfering RNA; siTFEB: Small interfering RNA targeting TFEB.

V8 and BTZ synergistically exhibit an antitumor effect in vivo

To validate the efficacy of combined treatment with V8 and BTZ against gastric cancer cells in vivo, MGC-803 cells were used to create a xenograft tumor model in nude mice. Combination of V8 and BTZ significantly inhibited the size and weight of subcutaneous transplanted tumors, compared with treatment with V8 or BTZ alone (Figure 6A-C). Expression of lysosome marker LAMP1 was also reduced in response to V8 treatment, suggesting lysosomal dysfunction occurred (Figure 6D and E). Ki67-positive cells were reduced, whereas caspase-3 cleavage was increased in tumor treated by combination of V8 and BTZ (Figure 6D, F and G). The other marker proteins involved in the apoptotic pathway were also upregulated in the combination treatment group (Figure 6A and B; Supplementary Figure 6). To determine whether the combined treatment resulted in chemical toxicity to normal tissue, the major organs were collected for pathological analysis. There was no significant toxic change in heart, liver, kidney tissues as indicating by HE staining between control and drug treatment groups (Figure 6H). The results showed that V8 combined with BTZ enhanced tumor suppression with good safety in vivo, implicating a potential strategy for gastric cancer therapy in clinic.

Figure 6 Effects of V8 and bortezomib on MGC-803 xenograft growth in vivo. A: Images of tumor xenograft from different treatment groups; B: Tumor volume was monitored for different treatment groups during experimental period; C: Tumor weight was calculated for different groups at the end of experiment; D-G: Immunohistochemistry was performed for analysis of LAMP1, Ki67 and cleaved-caspase-3 expression. Representative images (D) and quantified data (E-G) are shown (scale bar: 20 μm); H: Hematoxylin and eosin staining was performed in tissue sections from heart, liver and kidney. Representative images are shown (scale bar: 20 μm). ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. NS: No significance; BTZ: Bortezomib; AOD: Average optical density.

Combination of V8 and BTZ inhibits patient-derived tumor organoid growth

To further investigate the effect of V8 and BTZ on gastric cancer growth, we established a patient-derived tumor organoid model to test the sensitivity of gastric cancer to V8 and BTZ alone or in combination (Figure 7A). Gastric cancer organoids were sensitive to V8 treatment; only half of which survived when V8 concentration was up to 3 μM (Figure 7B). However, the range of drug exposure for lack of efficacy and increased toxicity is small, suggesting that V8 has a narrow therapeutic index. Similar to MGC-803 cells, gastric cancer organoids displayed resistance to high concentrations of BTZ (Figure 7C). The sensitivity of gastric cancer organoids to BTZ combined with V8 was further evaluated at the concentration more related to clinical dose. The combination significantly decreased the viability of organoids, compared with V8 or BTZ alone (Figure 7D-F). Our data showed potential application for clinical therapy of gastric cancer.

Figure 7 Combination of V8 and bortezomib suppresses patient-derived organoid growth. A: Scheme of patient-derived organoid culture and representative images (scale bar: 500 μm); B and C: Sensitivity of organoids to V8 and bortezomib (BTZ) was examined for 24 hours and 48 hours; D-F: Viability of organoids was evaluated in response to combination of V8 and BTZ treatment for 24 hours and 48 hours. Representative images (D) and heatmap of cell viability (E: 24 hours; F: 48 hours) are shown (scale bar: 500 μm). BTZ: Bortezomib.

DISCUSSION

MM cells are sensitive to low concentrations of BTZ (approximately 10 nM)[12,18]. However, most human gastric cancer cells are resistant to BTZ, even at a higher concentration[28,29]. Activation of protective autophagy could be responsible for solid tumor resistance to BTZ[33-36]. We also found that BTZ activated autophagy in gastric cancer cells. Surprisingly, autophagy inhibitors differentially affected the cytotoxicity of BTZ. Further investigation showed that BTZ could be trapped in lysosomes, which contributed to its resistance in gastric cancer cells. By combining a synthetic wogonin derivate, BTZ significantly suppresses cell viability of gastric cancer cells in vitro and in vivo. Thus, we showed a promising strategy for overcoming BTZ resistance in gastric cancer cells.

The role of lysosomes in drug resistance has been increasingly recognized. Although activation of the autophagy-lysosome pathway has been commonly attributed to protection of cancer cells from therapeutic agent-induced cell death, lysosomal sequestration of chemotherapeutic drugs with weakly basic property, such as daunorubicin, doxorubicin, sunitinib, imatinib, and sorafenib, also resulted in markedly reduced cytotoxicity and drug resistance[11,37,38]. Weak-base compounds are usually protonated in acidic lumina, which blocks their release due to decreased membrane permeability, known as ion trapping. Nevertheless, other mechanisms may also contribute to the lysosomotropism[39]. It is still not clear from the present study how BTZ is trapped in lysosomes, which needs further investigation. The cytotoxicity of lysosomotropic agents due to lysosomal dysfunction varies from micromolar to millimolar concentrations[40]. Lysosomal accumulation of some drugs alone is insufficient to induce lysosomal alkalization at nanomolar concentrations, but promotes TFEB-driven lysosomal biogenesis[30]. Consistent with previous study, our data showed that treatment of gastric cancer cells with BTZ at nanomolar concentrations did not deacidify lysosomes, but increased the number of lysosomes. However, there is still debate about lysosome-mediated drug resistance[41]. A more comprehensive understanding is still needed to solidify the current evidence.

Many lysosomotropic compounds exhibit antidepressant, antipsychotic, antihistamine, or antimalarial activity[40]. There is a growing recognition that targeting lysosomes might be a promising approach for treating various tumors. For example, the antimalarial hydroxychloroquine has been repurposed for diverse clinical oncology trials[42]. Recently, a novel chloroquine derivate, DC661, induced lysosomal lipid peroxidation (LLP) through targeting palmitoyl-protein thioesterase 1, resulting in tumor cell death[43]. Cell death driven by LLP, as a unique form of immunogenic cell death, facilitates immunotherapy of cancer. We previously synthesized a wogonin derivate V8 with antitumor potency[23]. The prodeath activity of V8 could be dependent on targeting lysosomal heat shock protein 70, as shown in T cell malignancy[22]. This could explain our results that intact lysosome function was required for V8-induced gastric cancer cell apoptosis. Considering the impairment of lysosomes caused by V8, it is not surprising that combination of V8 and BTZ induced integrated cellular stress encompassing ER stress, mitochondrial dysregulation, and ubiquitinated protein accumulation reflecting severe proteotoxic stress. While ROS contributed minimally, caspase-dependent apoptosis was central to cell death, as indicated by Z-VAD-FMK-mediated rescue. These results demonstrate that V8 and BTZ act synergistically to disrupt proteostasis and trigger apoptosis, therefore exerting an inhibitory effect on gastric cancer cells. Crucially, V8-induced lysosomal disruption not only releases sequestered BTZ but also blocks autophagic flux, preventing the clearance of ubiquitinated proteins and proteotoxic aggregates induced by BTZ. This dual action increasing proteotoxic burden while suppressing its clearance likely underlies the synergistic cytotoxicity of the combination treatment. Although it is widely accepted that apoptosis is not immunogenic, we found that apoptotic extracellular vesicles derived from tumor cells had an impact on immune cells through transferring proteins[44]. Hence, V8 together with BTZ might benefit remodeling of the tumor immune microenvironment in gastric cancer, which warrants further investigation.

Activation of TFEB pathway is considered to be a lysosomal stress response[32]. It is conceivable that V8 treatment upregulated TFEB-dependent autophagy gene expression. However, the autophagic flux was blocked by the lysosomal damage caused by V8. Although no lysosomal alkalinization occurred, BTZ also upregulated TFEB-responsive gene expression and activated autophagy. Our findings are in line with previous studies, in which proteasome inhibition and misfolded protein accumulation triggered TFEB nuclear translocation and autophagy activation[45,46]. Importantly, in our experimental system, V8-induced lysosomal dysfunction prevented autophagic degradation, rendering TFEB-driven autophagy incapable of mitigating severe proteotoxic stress. However, it is unknown whether compounds themselves such as MG-132, reversine, and AZ3146 used in those studies could be directly trapped into lysosome and then trigger stress response. In addition, Mlejnek et al[30] proposed that enhanced TFEB activity and lysosomal biogenesis was associated with G2/M cell cycle arrest, instead of lysosomal accumulation of chemotherapeutic agents. It is likely that BTZ-induced lysosomal biogenesis could be attributed to halting the cell cycle, although there is no evidence in the current study. We showed that constitutive activation of TFEB partially ameliorated apoptosis induced by V8 and BTZ alone or combination, probably depending on its role in lysosomal biogenesis. Nevertheless, TFEB also plays important roles in mitochondrial homeostasis, which can also respond to chemical-induced cell death[47]. The functional role of TFEB in cancer cell survival needs to be clarified in the presence of V8 and BTZ.

CONCLUSION

Our study showed that lysosomes mediated BTZ resistance in gastric cancer cells, and V8 induced lysosomal dysfunction, thereby increasing the accessibility of BTZ to proteasomes (Figure 8). Combination of V8 and BTZ inhibited gastric cancer cell growth in vitro, in vivo, and most prominently, in patient-derived tumor organoid models. This enhanced efficacy in patient-derived tumor organoids likely reflects their better preservation of original tumor heterogeneity and microenvironment, conferring higher proteasome dependency or specific pathway activation that renders them more sensitive to proteasome-inhibitor combinations. In contrast, long-term 2-dimensional cell lines may have lost some native tumor characteristics. These findings provide a strong rationale for developing this combination as a clinical therapeutic strategy.

Figure 8 Schematic diagram illustrating the mechanism by which V8 synergizes with bortezomib to induce cell death in gastric cancer cells. Bortezomib (BTZ) alone is sequestered in lysosomes, resulting in insufficient proteasome inhibition and mild apoptosis. V8 alone induces lysosomal damage and cell death via lysosomal membrane permeabilization and CTSB release. V8 disrupts lysosomes to release sequestered BTZ, enabling potent proteasome inhibition that triggers proteotoxic stress (ubiquitinated protein accumulation, endoplasmic reticulum stress, and mitochondrial stress), ultimately culminating in caspase-dependent apoptosis. BTZ: Bortezomib; TFEB: Transcription factor EB; CLEAR: Coordinated lysosomal expression and regulation; Bip: Immunoglobulin heavy chain binding protein; p-eIF2α: Phosphorylated eukaryotic translation initiation factor 2 alpha; Z-VAD-FMK: Benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone.

ACKNOWLEDGMENTS

The authors thank to Bao XQ (Pharmaceutical Animal Experimental Center, China Pharmaceutical University) for his kind help with animal studies.