Inhibition of Autophagy by Berbamine Hydrochloride Mitigates Tumor Immune Escape by Elevating MHC-I in Melanoma Cells

Abstract

Impaired tumor cell antigen presentation contributes significantly to immune evasion. This study identifies Berbamine hydrochloride (Ber), a compound derived from traditional Chinese medicine, as an effective inhibitor of autophagy that enhances antigen presentation in tumor cells. Ber increases MHC-I-mediated antigen presentation in melanoma cells, improving recognition and elimination by CD8⁺ T cells. Mutation of Atg4b, which blocks autophagy, also raises MHC-I levels on the cell surface, and further treatment with Ber under these conditions does not increase MHC-I, indicating Ber’s role in blocking autophagy to enhance MHC-I expression. Additionally, Ber treatment leads to the accumulation of autophagosomes, with elevated levels of LC3-II and p62, suggesting a disrupted autophagic flux. Fluorescence staining and co-localization analyses reveal that Ber likely inhibits lysosomal acidification without hindering autophagosome–lysosome fusion. Importantly, Ber treatment suppresses melanoma growth in mice and enhances CD8⁺ T cell infiltration, supporting its therapeutic potential. Our findings demonstrate that Ber disturbs late-stage autophagic flux through abnormal lysosomal acidification, enhancing MHC-I-mediated antigen presentation and curtailing tumor immune escape.

1. Introduction

Melanoma, a malignant skin cancer originating from melanocytes, is noted for its aggressive nature and poor prognosis. The global incidence of melanoma has been on the rise, with approximately 325,000 new cases reported worldwide in 2020 [1]. Additionally, lifestyle trends such as tanning and tattooing are contributing to a decreasing age of onset among populations [1]. Traditional treatment modalities, including surgery, chemotherapy, radiotherapy, and cytokine therapy, have demonstrated limited effectiveness against metastatic melanoma, leaving the five-year survival rate for these patients unsatisfactorily low [2]. This underscores an urgent demand for novel therapeutic interventions.

The landscape of melanoma treatment has been revolutionized by advancements in immunotherapy, significantly improving patient survival rates in recent years [3]. Tumor immunotherapy harnesses specific immune responses, particularly the activation of cytotoxic T cells (CD8⁺ T cells). These cells recognize and eliminate tumor cells that present antigens via major histocompatibility complex class I (MHC-I), thereby enhancing the efficacy of the treatment [4]. The antigen presentation by MHC-I molecules in cancer cells is critical in this process, and inefficiencies in this mechanism can facilitate tumor immune evasion [5]. Consequently, understanding the pathways that impair tumor antigen presentation is essential for the development of effective melanoma therapies.

Recent research has revealed that autophagy, a cellular degradation process, plays a crucial role in tumor immune evasion [6]. Autophagy not only controls the degradation of immune checkpoint proteins in cancer cells, but also affects antigen presentation and delivery by antigen-presenting cells, as well as modulates T cell activity [7,8]. Given these multifaceted roles, inhibiting autophagy could disrupt these tumor-protective mechanisms, potentially enhancing the immune system’s ability to recognize and destroy cancer cells. Moreover, reduced autophagy has been associated with an increase in reactive oxygen species within cells, leading to heightened susceptibility to apoptosis [9]. Therefore, targeting autophagy presents a promising strategy to amplify the effectiveness of cancer immunotherapy.

Currently, autophagy inhibitors are divided into two categories based on their mechanism of action. Early-stage inhibitors, such as 3-methyladenine (3-MA) and wortmannin, disrupt the initial formation of autophagosome membranes [10,11]. Late-stage inhibitors, including bafilomycin A1 (Baf) and chloroquine (CQ), obstruct the fusion of autophagosomes with lysosomes and inhibit lysosomal acidification [12,13]. Despite their potential, the clinical application of these inhibitors is often restricted due to their high toxicity [14]. Therefore, the development of safer, more effective autophagy inhibitors remains a critical clinical challenge.

Chinese herbal medicines, known for their vast array of bioactive properties, are increasingly recognized in drug development across various therapeutic areas. Berbamine hydrochloride (Ber), a natural monomeric compound extracted from Chinese medicinal herbs such as Coptis chinensis (Chinese goldthread) and Berberis spp. (barberry), exemplifies this potential. Historically noted for its potent antibacterial properties, Ber has shown considerable therapeutic effectiveness against diverse infectious diseases [15,16]. Clinically, it is predominantly utilized to elevate peripheral white blood cell counts [17]. Recent studies also highlight its anticancer capabilities [18], although the mechanisms underlying these effects warrant further exploration.

In our investigation, we identify Ber as a potent late-stage autophagy inhibitor that mitigates tumor immune evasion in melanoma. We demonstrate that Ber disrupts autophagy in melanoma cells by impairing lysosomal acidification, thereby enhancing MHC-I-mediated antigen presentation. This action significantly boosts the efficacy of CD8⁺ T cell-mediated tumor clearance, offering a promising avenue to reinforce anti-tumor immune responses in melanoma.

2. Materials and Methods

2.1. Cell Culture

B16-F10 (referred to as B16) melanoma cells were procured from the American Type Culture Collection (ATCC, Rockville, MD, USA), and A375 human melanoma cells were sourced from Wuhan Pricella Biotechnology (Wuhan, China). B16 cells stably expressing ovalbumin (OVA) were acquired from Beijing Crispr Biotechnology Co., Ltd. (Beijing, China). B16 cells were cultured in RPMI-1640 medium enriched with 10% fetal bovine serum (FBS), as reported in other publications [19,20], while A375 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% FBS. A375 cells stably expressing GFP-tagged LC3 (A375-GFP-LC3) were cultured under identical conditions to their wild-type counterparts. To induce autophagy via nutrient deprivation, A375-GFP-LC3 cells were washed with phosphate-buffered saline (PBS) and incubated in Hank’s Balanced Salt Solution (HBSS) for 6 h. All cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂.

2.2. Reagents and Antibodies

Berbamine hydrochloride (Ber, ≥99% purity, Catalog No. T2920) was sourced from TargetMol Chemicals Inc. (Shanghai, China). Autophagy inhibitors bafilomycin A1 (#S1413) and chloroquine (Catalog No. C6628) were obtained from Selleckchem (Houston, TX, USA). Primary antibodies targeting β-actin (#3700), LC3-I/II (#12741), p62 (#88588), cathepsin B (CatB, #31718), and cathepsin D (CatD, #2284) were purchased from Cell Signaling Technology (Boston, MA, USA). The primary antibody against HLA-A/B (A8754) was supplied by ABclonal (Wuhan, China), the anti-CD8a (#14-0081-85) antibody was purchased from Thermo Fisher Scientific (Waltham, MA, USA), and the H-2K^b (mouse MHC class I) antibody (#sc-59199) was obtained from Santa Cruz (Dallas, TX, USA). Secondary antibodies, HRP-conjugated anti-mouse IgG (#AS004), anti-rat IgG (#AS0028), and anti-rabbit IgG (#AS014) were procured from ABclonal (Wuhan, China); Alexa Fluor^® 594-conjugated anti-rat IgG was obtained from Abcam (#GR3438930-1, Cambridge, UK), and Alexa Fluor^® 594-conjugated anti-rabbit IgG (#8889) and Alexa Fluor^® 488-conjugated anti-rabbit IgG (#4412) were supplied by Cell Signaling Technology (Boston, MA, USA).

2.3. Western Blot Analysis

Post-treatment, cells were lysed using a buffer supplemented with protease inhibitors. Protein concentrations were determined using the BCA Protein Assay Kit (#MA0082, Meilunbio, Dalian, China). The samples (20 µg protein each) were separated by SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% skim milk in TBST, incubated overnight at 4 °C with primary antibodies (dilution 1:1500) and then with horseradish peroxidase-conjugated secondary antibodies for one hour at room temperature. Protein bands were visualized using the Tanon™ 5200CE Chemi-Image System (Tanon, Shanghai, China).

2.4. In Vitro Cytotoxicity Assay

OT-I mice, harboring T cell receptors specific to OVA_257-264 presented by H-2K^b, were sourced from GENEANDPEASE (Yangzhou, China). Their spleens were harvested, mechanically dissociated, and lymphocytes were isolated using a lymphocyte separation solution (#7211011, Dakewe, Shenzhen, China). CD8⁺ T cells were enriched by incubating with mouse CD8a (Ly-2) MicroBeads (#130-117-044, Miltenyi Biotec, Bergisch Gladbach, Germany) and MACS buffer (#130-091-222-1, Miltenyi Biotec) at 4 °C for 10 min, followed by separation using a magnetic column. The cells were subsequently resuspended in complete medium enriched with IL-2 (#212-12-100, Peprotech, Cranbury, NJ, USA) for further experiments.

B16-OVA cells were seeded in 12-well plates overnight and subsequently treated with varying concentrations of Ber. After treatment, the drug was washed out, and the cells were co-cultured with CD8⁺ T cells derived from OT-I mice (referred to as OT-I T cells) for 24 h. Post-co-culture, supernatants were collected for IFN-γ quantification using an ELISA kit (#RK00019, ABclonal, Wuhan, China). Simultaneously, co-cultured cells were washed with PBS to remove OT-I T cells. The remaining B16-OVA cells were then fixed with 4% paraformaldehyde, washed twice with PBS, and stained with crystal violet for 10 min. After extensive rinsing with PBS, the viability of tumor cells was quantified by measuring the optical density at 570 nm following crystal violet staining. To assess T cell-mediated cytotoxicity following Ber treatment, co-cultured OT-I T cells and Ber-pre-treated B16-OVA cells were harvested and subjected to dual staining with Fixable Viability Stain 620 (#553142, BD Biosciences, San Jose, CA, USA) and APC-conjugated anti-mouse CD8a (#100712, Biolegend, San Diego, CA, USA). Flow cytometry analysis was performed to identify the apoptotic B16-OVA cells, which were characterized as CD8a-negative and FVS620-positive, indicative of T cell-mediated tumor cell killing.

2.5. Flow Cytometry Analysis

Murine or human tumor cells, treated with Ber for designated time intervals, were harvested by trypsinization and washed with PBS containing 2% FBS. For immunophenotyping, the cells were stained in the dark at room temperature for 20 min using the following antibodies: Pacific Blue™ anti-mouse H-2K^b (#116514, BioLegend, San Diego, CA, USA), FITC anti-mouse H-2K^b (#553569, BD Biosciences, San Jose, CA, USA), and the OVA257-264 peptide bound to H-2K^b (#141605, BioLegend). After staining, the cells were washed twice, resuspended in a buffer, filtered through a 70 µm nylon mesh, and immediately analyzed using a BD LSR Fortessa flow cytometer. Data analysis was conducted using Flow Jo software (version. 10.8.1; Tree Star Inc., Ashland, OR, USA).

2.6. Immunofluorescence Assay

Cells were plated on coverslips in 12-well plates and treated with Ber for 24 h. Following a PBS wash, the cells were fixed with 4% paraformaldehyde for 20 min at room temperature. Blocking was performed for two hours with 5% fetal bovine serum in PBS. The cells were then incubated overnight at 4 °C with a primary antibody specific to HLA-A/B or LC3-I/II. After washing with PBS, the cells were incubated with Alexa Fluor^® 594-conjugated anti-rabbit IgG or Alexa Fluor^® 488-conjugated anti-rabbit IgG for one hour at room temperature, followed by staining with Hoechst 33342 (5 µg/mL) for 10 min. Slides were mounted using a Prolong™ Diamond Antifade Mountant (#P36970, Invitrogen, Carlsbad, CA, USA) and imaged with an LSM 800 confocal microscope (Carl Zeiss, Jena, Germany). For immunofluorescence staining of paraffin sections, sections underwent antigen retrieval and blocking prior to overnight incubation with an antibody against CD8a. They were then incubated with Alexa Fluor^® 594-conjugated anti-rat IgG for one hour at room temperature, counterstained with DAPI, and analyzed with a confocal microscope.

2.7. Lentivirus Production and Infection

Lentiviral vectors were transfected into HEK293FT cells using Polyethylenimine Linear (PEI) MW40000 (#40816ES02, Yeasen, Shanghai, China) in Opti-MEM™ (#31985062, Gibco, Grand Island, NY, USA). For lentivirus production, HEK293FT cells were co-transfected with the lentiviral vector and packaging plasmids psPAX2 and pMD2.G at a 1:3:4 ratio. Six hours post-transfection, the medium was replaced with DMEM supplemented with 10% FBS. Viral supernatants were harvested 48 h later, filtered through a 0.22 μm pore-size filter, and used for infecting cells in DMEM supplemented with 10% FBS. GFP-LC3 plasmids were provided by Professor William KK Wu (The Chinese University of Hong Kong), and the pSLIK_Hyg_mTurquoise2_Atg4bC74A plasmids were sourced from Alec Kimmelman’s lab via Addgene (#161733, Watertown, MA, USA). The hLAMP1-mCherry construct (#VB900162-5529yba) was designed and purchased from VectorBuilder (Guangzhou, China).

2.8. mCherry-GFP-LC3B Transfection

The pBABE-puro mCherry-GFP-LC3B plasmid (Addgene, #22418) was a gift from Jayanta Debnath [21]. Cells were seeded onto glass coverslips in 12-well plates and transiently transfected with 0.5 µg of the plasmid. After 24 h, the cells were treated with Ber, fixed with 4% paraformaldehyde, and evaluated for autophagy flux using an LSM 800 confocal microscope (Carl Zeiss, Jena, Germany). The number of puncta per cell was counted and compared by researchers who were blinded to the grouping information.

2.9. RNA Isolation and Quantitative RT-PCR

Total RNA was extracted using TRIzol reagent (#15596018CN, Thermo Fisher Scientific) following the manufacturer’s guidelines. The RNA concentration was measured, and samples were reverse transcribed into cDNA using an Evo M-MLV RT Kit (#AG11706, Accurate Biology, Changsha, China). mRNA levels were quantified using SYBR-Green qPCR Master Mix (#AG11740, Accurate Biology). The sequences of the qRT-PCR primers can be found in Supplementary Table S1.

2.10. LysoTracker Staining

A375 cells were seeded into a confocal dish and incubated for 24 h. On the second day, drug treatment was administered according to the experimental design. After treatment, the medium was discarded, and the cells were washed twice with PBS. Lysosomes were then stained using LysoTracker Red DND-99 (50 nM, L7528; Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Images were captured using a laser scanning confocal microscope (LSM 800; Carl Zeiss, Jena, Germany) and analyzed using ImageJ software (Version 1.4.3; NIH, Bethesda, MA, USA).

2.11. Autophagosome and Lysosome Colocalization

A375 cells stably expressing GFP-LC3 were cultured in confocal dishes and treated with Ber and Chloroquine (CQ) for 24 h. Post-treatment, the cells were washed twice with PBS and stained with LysoBrite™ Red for 20 min at 37 °C in DMEM to label lysosomes. After staining, the cells were washed again with PBS and imaged using an LSM 800 confocal microscope (Carl Zeiss, Jena, Germany).

2.12. Electron Microscopy

Cells were rinsed twice with PBS and collected into a 15 mL centrifuge tube. The cell suspension was centrifuged at 1500–3000 rpm for 5 min. The cell pellets were then fixed with 2.5% glutaraldehyde in the dark at room temperature for 30 min. The fixed cells were mounted on copper grids and examined using a transmission electron microscope.

2.13. Immunohistochemistry

Tumor tissues from mice were fixed in 4% paraformaldehyde overnight, embedded in paraffin, and sectioned into 4 µm slices. The sections were deparaffinized, rehydrated, and subjected to antigen retrieval in a boiling citrate buffer (pH 6.0). After blocking with 5% bovine serum albumin, the sections were incubated overnight at 4 °C with anti-CD8a antibody. This was followed by a one-hour incubation with HRP-conjugated anti-rat IgG at a 1:200 dilution at room temperature. The stained sections were then imaged using a pathology slide scanner.

2.14. In Vivo Analysis

The experiments involving mice were approved by the Animal Ethics Committee at Guangzhou University of Chinese Medicine (Project No. 20240428018). Eight-week-old C57BL/6 mice were purchased from the Guangdong Provincial Medical Laboratory Animal Center and acclimated in a specific pathogen-free (SPF) facility. They were provided with ad libitum access to sterilized water and food. B16 cells (2 × 10⁶ cells in 100 µL PBS) were subcutaneously injected into the right flank of the mice. The mice were then randomly divided into three groups (n = 5 per group) and subjected to daily intraperitoneal injections with either vehicle (saline solution), 10 mg/kg Ber, or 20 mg/kg Ber. When the tumor volume approached nearly 800 mm³, the mice were anesthetized, and the tumors were dissected and weighed.

2.15. Cell Counting Kit-8 (CCK-8) Assay

To investigate the effect on the proliferation of B16 cells, the cells were seeded at a density of 10,000 cells per well in a 96-well plate. Then, the corresponding drugs were added to the cells according to the experimental protocol. Cell Counting Kit-8 (MA0218, Meilunbio, Dalian, China) was used to examine the proliferation of B16 cells, following the manufacturer’s protocol.

2.16. Statistical Analysis

Statistical analyses were conducted using GraphPad Prism version 6 (GraphPad Software, San Diego, CA, USA). The data are presented as the mean ± standard deviation (SD) or standard error of the mean (SEM). Differences between groups were assessed using Student’s t-test, one-way ANOVA with Dunnett’s or Tukey’s multiple comparisons test, or two-way ANOVA with Tukey’s multiple comparisons test. Statistical significance was defined as * p < 0.05, ** p < 0.01, and *** p < 0.001; ns indicates not significant.

3. Results

3.1. Ber Enhances MHC-I-Mediated Antigen Presentation in Melanoma Cells

Ber is a natural monomer derived from traditional Chinese medicinal herbs, such as Coptis chinensis (Chinese goldthread) and Berberis spp. (barberry). The molecular structure of Ber is depicted in Figure 1A. The downregulation of MHC class I molecules on tumor cells contributes significantly to immune evasion [22]. To assess the effects of Ber on MHC-I expression, we treated the human melanoma cell line A375, which expresses HLA-A/B as MHC-I, and the mouse melanoma cell line B16, which expresses H-2K^b as MHC-I, with various concentrations of Ber for 24 h. Flow cytometric analysis demonstrated that Ber treatment increased the surface expression of MHC-I in a concentration-dependent manner in B16 cells (Figure 1B). Similarly, immunofluorescence staining showed a significant increase in HLA-A/B expression in A375 cells post-Ber treatment (Figure 1C). Western blot analysis further confirmed that Ber treatment resulted in a concentration-dependent increase in the intracellular abundance of MHC-I proteins in both A375 and B16 cell lines (Figure 1D). To evaluate the impact of Ber on antigen presentation efficiency, we analyzed the presentation of the fluorescently labeled artificial antigen complex H-2K^b-SIINFEKL in B16-OVA cells. SIINFEKL is a peptide derived from ovalbumin (OVA). After treating B16-OVA cells with varying concentrations of Ber for 24 h, we observed enhanced expression of the surface H-2K^b-SIINFEKL complex via flow cytometry (Figure 1E). These findings suggest that Ber effectively increases MHC-I-mediated antigen presentation by upregulating surface and intracellular MHC-I levels in melanoma cells.

3.2. Ber Enhances CD8⁺ T Cell-Mediated Cytotoxicity against Melanoma Cells

CD8⁺ T cells target and eliminate tumor cells presenting specific antigens. To determine whether Ber augments the cytolytic activity of T cells through enhanced antigen presentation, B16-OVA melanoma cells were treated with varying concentrations of Ber for 24 h. These cells were then co-cultured with CD8⁺ T cells from OT-I mice, which recognize the ovalbumin (OVA) antigen, for an additional 24 h (Figure 2A). Crystal violet staining revealed that the cytolytic activity of CD8⁺ T cells increased in a concentration-dependent manner with Ber treatment (Figure 2B). Interferon-gamma (IFN-γ), a critical cytokine for tumor cell recognition and elimination by CD8⁺ T cells, was measured in the co-culture supernatants. Ber treatment enhanced IFN-γ production dose-dependently (Figure 2C). Additionally, flow cytometry analysis was performed to quantify apoptotic tumor cells within the co-culture. The cells were stained with CD8 and FVS620, a dye that penetrates dead cells and binds to lipid membranes, emitting a strong red fluorescence. A progressive increase in the proportion of apoptotic tumor cells (CD8⁻/FVS620⁺) with escalating doses of Ber was observed (Figure 2D). To exclude the influence of Ber on cellular viability, we conducted a CCK8 assay and confirmed that Ber (0, 0.2, 1, 5 µM) did not impact the viability of B16 cells (Figure S2). These findings indicate that Ber enhances MHC-I-mediated antigen presentation, thereby improving the efficacy of CD8⁺ T cells in recognizing and eliminating melanoma cells.

3.3. Ber Increases MHC-I Levels in Melanoma Cells by Inhibiting Autophagy

While investigating the mechanisms underlying the increase in MHC-I expression induced by Ber, we first determined whether this was due to enhanced transcription. Our results showed that Ber had no significant effect on MHC-I transcription in either A375 or B16 cells (Figure S1). This led us to hypothesize that Ber enhances MHC-I-mediated tumor antigen presentation through the inhibition of autophagy. To test this hypothesis, we utilized a doxycycline (Dox)-inducible Atg4b mutation in B16 cells, where Atg4b plays a critical role in autophagosome formation and degradation. Western blot analysis showed a significant, dose-dependent increase in MHC-I (H-2K^b) levels following 24 h of autophagy inhibition with Dox (Figure 3A). Flow cytometry further confirmed a significant increase in surface MHC-I (H-2K^b) levels after blocking autophagy with the mutated Atg4b and higher Dox concentrations (Figure 3B).

Moreover, when autophagy was inhibited by mutating Atg4b and cells were subsequently treated with Ber, there was no further increase in MHC-I (H-2K^b) levels (Figure 3C,D). Additionally, analysis of fluorescently labeled H-2K^b-SIINFEKL demonstrated increased surface expression following treatment with varying concentrations of Dox, whereas simultaneous treatment with Ber and Dox did not further enhance the levels of H-2K^b-SIINFEKL (Figure 3E,F). These findings suggest that Ber’s enhancement of MHC-I expression and subsequent improvement in antigen presentation in melanoma cells primarily operates through the inhibition of autophagy.

3.4. Ber Inhibits Autophagic Flux in Melanoma Cells

In our studies, we noted that Ber elevated MHC-I expression in tumor cells by impeding autophagy. To assess Ber’s impact on autophagy, we first quantified LC3-positive puncta, indicative of autophagosomes, in A375 cells transfected with GFP-LC3. Treatment with Ber (5 µM for 24 h) increased the number of LC3-positive puncta, an effect comparable to that induced by Hank’s Balanced Salt Solution (HBSS, commonly used to induce autophagy via starvation) and the autophagy inhibitor bafilomycin A1 (Baf), suggesting that Ber modulates autophagic processes (Figure 4A). We further investigated the conversion of LC3. During autophagy, cytoplasmic LC3-I converts into membrane-bound LC3-II, which associates with autophagosome membranes and is subsequently degraded in autolysosomes [23]. We observed that LC3-II levels in A375 and B16 cells increased in a time- and dose-dependent manner following Ber treatment (Figure 4B,C), indicating an accumulation of autophagosomes.

To differentiate whether this accumulation was due to increased formation or decreased degradation of autophagosomes, we measured levels of p62. p62 binds ubiquitinated proteins and forms complexes with LC3-II that are degraded in autolysosomes; thus, elevated p62 levels typically suggest disrupted autophagy [24]. We found that Ber treatment caused a concentration-dependent increase in p62 levels (Figure 4B,C), implying that the augmentation in autophagosomes was due to impaired degradation, not increased formation. In alignment with the increases in LC3-II and p62 induced by Ber, we documented a significant rise in LC3-positive puncta in A375 cells transfected with GFP-LC3. Given our previous observations, this likely results from the accumulation of autophagosomes attributable to hindered autophagic degradation. While HBSS treatment alone increased the number of LC3-positive puncta through enhanced autophagosome formation and efficient autophagic flux, the addition of Ber led to a further increase in these puncta (Figure 4D). This finding supports the notion that Ber disrupts the otherwise smooth autophagic flux, leading to an accumulation of undegraded autophagosomes.

3.5. Ber Inhibits Late-Stage Autophagy in Melanoma Cells by Suppressing Lysosomal Acidification

To investigate whether Ber affects the degradation stage of autophagy, we transfected A375 cells with an mCherry-GFP-LC3B tandem reporter, which differentiates autophagosomes from autolysosomes based on their distinct fluorescence. Typically, autophagy induction by starvation (HBSS) increases red fluorescence due to GFP quenching in acidic environments, while disruptions in autophagosome–lysosome fusion or lysosomal function result in persistent yellow fluorescence. Treatment with Ber (5 µM, 24 h) significantly increased the presence of yellow puncta, similar to the effect of the autophagy inhibitor chloroquine (CQ), indicating an impairment in the late stages of autophagic flux (Figure 5A). To determine whether Ber inhibits autophagic flux by affecting autophagosome–lysosome fusion or by impairing autolysosomal degradation, we analyzed the colocalization of GFP-LC3 (autophagosomes) and LysoBrite™ Red (a marker for lysosomes or autolysosomes) in GFP-LC3-transfected A375 cells. CQ treatment led to the accumulation of GFP-LC3 puncta with minimal colocalization with lysosomes, indicating disrupted fusion. In contrast, Ber treatment resulted in extensive colocalization, producing numerous yellow puncta (Figure 5B), suggesting that autophagosomes successfully fused with lysosomes.

To further confirm this, we transfected A375 cells with the hLAMP1-mCherry plasmid to directly observe the colocalization of LAMP1 (a lysosomal marker) and LC3 (an autophagosomal marker). Unlike CQ treatment, Ber treatment led to significant colocalization of LAMP1 and LC3 (Figure 5C), further supporting intact autophagosome–lysosome fusion. Additionally, LysoTracker staining, a well-established method for detecting lysosomal acidification, demonstrated that Ber significantly reduced lysosomal acidity (Figure 5D). Ber also decreased the levels of mature lysosomal enzymes such as cathepsin B and cathepsin D, both of which depend on normal lysosomal acidification (Figure 5E). Moreover, electron microscopy of Ber-treated A375 cells revealed the presence of autolysosomes formed after autophagosome–lysosome fusion (Figure 5F). These findings provide strong evidence that Ber does not inhibit autophagosome–lysosome fusion but rather suppresses lysosomal acidification, thereby inhibiting late-stage autophagy in melanoma cells.

3.6. Ber Suppresses Melanoma Tumor Growth in Mice by Enhancing CD8⁺ T Cell Infiltration

To evaluate the effects of Ber on melanoma tumor growth, B16 melanoma cells were subcutaneously injected into the right axillary region of C57BL/6 mice to establish subcutaneous tumors. The mice were randomly assigned into three groups: Ber-treated at 0 mg/kg, Ber-treated at 10 mg/kg, and Ber-treated at 20 mg/kg. Tumor volume was measured daily (n = 5 per group) (Figure 6B). After 14 days, the animals were euthanized, and tumors were harvested and weighed (Figure 6A,C). Notably, a significant suppression of tumor growth was observed in both Ber-treated groups. Immunohistochemical and immunofluorescent analyses of CD8 staining in subcutaneous B16 tumors from mice were conducted to assess the infiltration of CD8⁺ T cells within the tumor tissues. The results demonstrated that Ber treatment significantly increased CD8⁺ T cell infiltration in melanoma tumors (Figure 6D,E). These findings demonstrate that Ber not only inhibits melanoma progression but also enhances the immune response within the tumor microenvironment, potentially through the recruitment and activation of cytotoxic T cells.

4. Discussion

We report that Ber upregulates MHC-I expression on the surface of melanoma cells, thereby enhancing antigen presentation and the subsequent recognition and elimination of tumor cells by CD8⁺ T cells, circumventing tumor immune escape. Given the known role of autophagy in tumor immune evasion [25], we hypothesized that Ber might augment MHC-I protein expression by inhibiting autophagy, thus boosting antigen presentation. Our findings confirm this hypothesis, showing that autophagy inhibition raises MHC-I protein levels on melanoma cells, and further increases in MHC-I expression are not observed with Ber treatment following Atg4b mutation. This lack of an additive effect suggests that Ber’s primary mechanism involves autophagy inhibition.

Further investigation revealed that Ber inhibits autophagy by suppressing lysosomal acidification within tumor cells. MHC (major histocompatibility complex) molecules play a crucial role in enabling T cells to specifically recognize and target cells. We focus particularly on MHC-I, as CD8⁺ T cells recognize antigens presented by MHC-I on the surface of tumor cells, thereby initiating immune responses [26,27]. A reduction in surface MHC-I expression can allow tumor cells to evade recognition by CD8⁺ T cells, facilitating immune evasion. Previous studies corroborate our findings; for instance, Angel M. Garcia-Lora et al. demonstrated that MHC-I downregulation enhances melanoma cell carcinogenicity, proliferation, migration, and invasion in vivo and in vitro [26]. Similarly, Xiaowei Liu et al. reported a significant association between MHC-I antigen presentation loss in melanoma cells and resistance to PD-1 blockade [27]. Thus, enhancing MHC-I expression on tumor cells is crucial for improving the efficacy of melanoma therapies.

In our study, we treated two melanoma cell lines with varying concentrations of Ber and observed a significant increase in surface MHC-I expression (Figure 1B). We also noted a rise in total cellular MHC-I protein levels (Figure 1D). Notably, Ber did not affect MHC-I transcription in either melanoma cell line (Figure S1), leading us to speculate that Ber modulates MHC-I degradation pathways. Proteins may be degraded through the proteasome pathway or the autophagy–lysosome pathway. Interestingly, when autophagy was inhibited via a (Dox)-inducible Atg4b mutation and the cells were subsequently treated with Ber, there was no further increase in MHC-I (H-2K^b) levels (Figure 3C,D), and H-2K^b-SIINFEKL presentation was not additionally enhanced (Figure 3E,F). These findings suggest that Ber primarily enhances MHC-I expression and antigen presentation in melanoma cells through the inhibition of autophagy.

The 257-264 peptide of the ovalbumin (OVA) protein is known to be presented by MHC-I. In OVA-expressing B16 cells, it forms complexes with MHC-I on the cell surface. These complexes can be specifically recognized and bound by the antibody, marking cells that have undergone OVA antigen presentation. Flow cytometry analysis showed that Ber treatment increased antigen presentation levels in a concentration-dependent manner (Figure 1E). Increasing antigen presentation levels theoretically enhances the specific killing by CD8⁺ T cells. For example, Arul M. Chinnaiyan et al. demonstrated that augmenting tumor-specific MHC-I expression increases the number of functional CD8⁺ T cells within the tumor and slows tumor progression in several syngeneic mouse models [28]. The T cell receptor (TCR) on CD8⁺ T cells recognizes and binds to the tumor antigen-MHC-I complex on the cell surface, triggering the release of cytotoxic substances such as perforin, tumor necrosis factor, and interferon to kill tumor cells [29]. We further validated these findings by co-culturing Ber-treated B16-OVA cells with CD8⁺ T cells from OT-I mice, which recognize the OVA protein’s 257-264 peptide presented by MHC-I. Post-treatment, we observed an enhanced killing capacity of T cells (Figure 2B,D), and ELISA assays confirmed a significant increase in IFN-γ release in the co-culture system, indicating CD8⁺ T cell activation. In vivo experiments also confirmed that Ber significantly increased CD8⁺ T cell infiltration in melanoma tumors and inhibited tumor growth (Figure 6), substantiating Ber’s potential as an immunotherapeutic agent in melanoma treatment.

Our hypothesis posits that Ber enhances MHC-I expression in tumor cells by inhibiting autophagy, a process increasingly recognized as a pivotal factor in tumor immune evasion [25,30]. For instance, Ravi K. Amaravadi et al. observed that stage IV melanoma patients exhibiting poor responses to dacarbazine and sorafenib chemotherapy had elevated autophagy levels in tumor cells [31]. Moreover, research indicates that melanoma resistance to chemotherapy may be associated with autophagy induction triggered by Wnt5A expression [32]. Autophagy typically progresses from the formation of a pre-autophagosomal structure (PAS) upon sensing cellular stress, to the recruitment and assembly of autophagy-related proteins (ATGs) that form autophagosomes. These autophagosomes eventually fuse with lysosomes to create autolysosomes, where their contents are degraded by enzymes [33]. Initially, autophagy may suppress tumor formation by mitigating oncogenic stressors such as chronic tissue damage and genomic instability [34,35]. However, in advanced tumors, autophagy may help cancer cells survive under adverse conditions like hypoxia and nutrient deprivation [36,37]. Given that most cancers are diagnosed at advanced stages, targeting autophagy could provide therapeutic benefits.

Ber has been reported as an autophagy inhibitor, with Fu et al. showing that Ber suppresses the accumulation of autophagosomes in breast cancer cells by upregulating BNIP3 and blocking autophagosome–lysosome fusion [38]. In contrast, our study reveals that in melanoma cells, Ber operates through a distinct mechanism—specifically, by inhibiting lysosomal acidification, which in turn suppresses late-stage autophagy. Our LysoTracker staining, a well-established method for detecting lysosomal acidification, demonstrated that Ber treatment significantly reduced lysosomal acidity (Figure 5D). Additionally, Ber decreased the levels of mature lysosomal enzymes such as cathepsin B and cathepsin D, both of which are dependent on normal lysosomal acidification (Figure 5E). These findings strongly suggest that Ber inhibits autophagy by suppressing lysosomal acidification, thereby impairing the final stage of autophagic flux. To further elucidate whether Ber inhibits autophagic flux by affecting autophagosome–lysosome fusion, we conducted a co-localization analysis using GFP-LC3 and LysoBrite™ Red, a dye less sensitive to acidity changes than LysoTracker. Our analysis revealed that GFP-LC3-positive puncta in Ber-treated cells largely co-localized with lysosomes (Figure 5B). Additionally, we transfected A375 melanoma cells with the hLAMP1-mCherry plasmid to mark lysosomes with red fluorescence, while autophagosomes were marked with green fluorescence via immunofluorescence staining of LC3. Unlike chloroquine (CQ) treatment, Ber treatment resulted in significant co-localization of LAMP1 (lysosomes) and LC3 (autophagosomes) (Figure 5C). Furthermore, electron microscopy of Ber-treated A375 cells revealed the presence of autolysosomes, formed after the fusion of autophagosomes and lysosomes (Figure 5F). These results provide clear evidence that Ber does not prevent autophagosome–lysosome fusion but rather inhibits lysosomal acidification, thereby suppressing late-stage autophagy in melanoma cells. While our findings differ from those of Fu et al. [38], this discrepancy may be attributed to the distinct cellular contexts of different cancer types. Nevertheless, our study significantly advances the understanding of Ber’s mechanism in melanoma cells and supports the broader conclusion that Ber inhibits late-stage autophagy.

Recent interest in the therapeutic potential of natural products for drug development has grown significantly. Ber, derived from the Chinese medicinal herbs Coptis chinensis (Chinese goldthread) and Berberis spp. (barberry), is primarily known for its anti-inflammatory properties [15,39]. Its hydrochloride form, particularly, has been explored for anticancer activities. Notable studies include Han C et al., who demonstrated that Ber modulates the ROS/NF-κB signaling pathway to exert anti-tumor effects in bladder cancer [40]; Bingren Hu et al., who highlighted its efficacy against pancreatic cancer [41]; Parhi et al., who observed inhibitory effects on the growth of B16 melanoma in a C57BL/6 mouse model [42]; and Fu et al., who reported that Ber suppresses the accumulation of autophagosomes in breast cancer cells [38]. Our study adds to this field by demonstrating that Ber, as a late-stage autophagy inhibitor, suppresses autophagy in melanoma cells by inhibiting lysosomal acidification. This inhibition significantly enhances MHC-I-mediated antigen presentation, thereby boosting the specific immune killing of tumors by CD8⁺ T cells.

Currently, only chloroquine and its derivative hydroxychloroquine are FDA-approved for this purpose, primarily due to safety concerns with other compounds [43]. Given the historical use of Ber and its established safety profile [44], it emerges as a promising candidate for the development of safer and more effective autophagy inhibitors. In a therapeutic landscape where options for clinical autophagy inhibitors are scarce, Ber could potentially augment tumor cell eradication when used alongside conventional anticancer drugs through its modulation of autophagy. In summary, Ber’s ability to inhibit lysosomal acidification disrupts autophagic flux in melanoma cells. This disruption enhances MHC-I-mediated antigen presentation, thereby improving the efficacy of CD8⁺ T cell-mediated tumor cell destruction. This mechanism highlights Ber’s potential as a valuable adjunct in melanoma therapy and underscores the broader applicability of natural products in medical research.

5. Conclusions

Our research demonstrates that Ber effectively inhibits late-stage autophagy by targeting lysosomal acidification, enhancing MHC-I-mediated antigen presentation in melanoma cells. This improvement in antigen presentation facilitates better recognition and elimination of tumor cells by CD8⁺ T cells. These findings suggest that Ber has potential as an adjuvant therapy in melanoma treatment.