The following reagents were used: restriction enzymes (BamHI, HindIII, SmaI), DNA marker, and protein markers (MBI Ferments, Burlington, Ontario, CA); EndoFree Plasmid Maxi Kits, IPTG, and Ni-NTA agarose (QIAGEN, Hilden, Germany); RPMI-1640 medium, FBS, penicillin, and streptomycin (Gibco, USA); Lipofectamine™ 3000 reagent (Thermo, USA); DeadEnd™ Fluorometric TUNEL System (Promega, USA); Annexin V-FITC/PI apoptosis kit (MultiSciences, China); LDH Release Assay Kit, CellTiter-Lumi™ Luminescent Cell Viability Assay Kit (Beyotime, China); human IL-18, IL-1β, and HMGB-1 ELISA kits (Elabscience, China); and anti-FLAG Affinity gel purified kit (Sangon Biotech, China). Primary antibodies used included: anti-His tag (66005-1-Ig, Proteintech); anti-GSDME, anti-GrB, anti-Bax, anti-Caspase-3, and anti-FLAG (Abcam, ab215191, ab208586, ab32503, ab32351, ab205606); anti-Caspase-8, and anti-Snail (Cell Signaling Technology, #9496, #3879); anti-GAPDH (Good Here, AB-P-R 001); anti-HPV16E7, anti-E-Cadherin, N-cadherin, anti-Vimentin, and anti-Bcl2 (Santa Cruz, sc-6981, sc-8426, sc-53488 sc-6260, sc-56018). Secondary antibodies included HRP-labelled goat anti-rabbit IgG, HRP-labelled goat anti-mouse IgG, Cy3-labelled goat anti-mouse IgG, and FITC-labelled goat anti-rabbit IgG (Beyotime, A0208, A0216, A0521, A0562). The HPV16E7 protein without the His tag was prepared in our laboratory.

Plasmids containing coding sequences, including pET21a (+)/Z_HPV16E7-GrB, pET21a (+)/Z_HPV16E7, and pET21a (+)/Z_WT-GrB (wild-type Z_WT affibody without affinity screening, used as an untargeted control), were constructed and maintained in our laboratory. The plasmid pCMV-3×FLAG-GSDME (human)-Neo (P68941) was sourced from MiaoLingPlasmid, China. The following human cervical cancer cell lines were used: SiHa (HPV16-positive, ATCC HTB-35), CaSki (HPV16-positive, ATCC CRM-CRL-1550), HeLa (HPV18-positive, ATCC CCL-2.1, as the HPV type control), and C33A (HPV-negative, ATCC HTB-31, as the HPV-free control). These cell lines were purchased from the Cell Bank of the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, and cultured in RPMI-1640 medium supplemented with 100 mg/L penicillin and streptomycin and 10% fetal bovine serum (FBS) at 37°C in a 5% CO₂ humidified atmosphere.

We first systematically optimized its amino acid sequence (GenBank No. AAA75490.1) through two critical modifications: (i) mutation of key residues to prevent protease inhibition, and (ii) replacement of polar amino acids with nonpolar counterparts to reduce nonspecific adsorption. The engineered GrB gene was subsequently synthesized and directionally cloned into the pcDNA3.1(+) vector using HindIII and BamH I restriction sites. For functional analysis, we performed transient transfection of SiHa cells in 6-well plates using 1 μg of the recombinant pcDNA3.1/GrB plasmid per well with Lipofectamine™ 3000, with protein expression confirmed after 24-hour incubation by both Western blot and indirect immunofluorescence. Finally, to fully characterize GrB’s biological effects, we implemented a multi-modal analytical approach: (1) quantitative assessment of apoptosis induction using flow cytometry combined with TUNEL assay, (2) detection of GSDME cleavage (a key executioner of pyroptosis) by Western blot, followed by precise quantification of protein band intensities using ImageJ software to ensure rigorous data analysis.

The immunoaffitoxin Z_HPV16E7-GrB was engineered in our laboratory (25) as a fusion protein comprising three key components: (1) an HPV16E7-specific affibody (Z_HPV16E7), (2) human granzyme B (GrB), and (3) a flexible peptide linker (Gly₄Ser)₃ connecting these domains, with an N-terminal 6× His tag to facilitate purification. For recombinant protein expression, we transformed E. coli BL21(DE3) competent cells with three plasmid constructs: pET21a (+)/Z_HPV16E7-GrB, pET21a (+)/Z_HPV16E7, and pET21a (+)/Z_WT-GrB). Each transformation used 100ng plasmid DNA per 50 μL aliquot of competent cells. Following transformation, positive clones were selected and expanded in LB medium. Protein expression was then induced with 1 mM IPTG for 8 hours at 37°C, and the target proteins were purified via Ni²⁺-chelated affinity chromatography. The purified proteins were verified by both SDS-PAGE and Western blot analysis. Finally, the three-dimensional structure of the immunoaffitoxin was predicted using the GalaxyWEB server (http://galaxy.seoklab.org/).

The binding affinity and targeting specificity of Z_HPV16E7-GrB were evaluated using two approaches: an ELISA assay to assess its affinity for the HPV16E7 protein and an indirect immunofluorescence assay to determine its targeting ability toward HPV16-positive cells. For the ELISA assay, high-adhesion 96-well flat-bottom microtiter plates were first coated overnight at 4°C with 100 μL of HPV16E7 protein (10 μg/mL, His-tag-free), followed by blocking with 5% skim milk in PBST at 37°C for 2 hours. The wells were then incubated overnight at 4°C with 300 μg/mL of Z_HPV16E7-GrB, Z_HPV16E7, or Z_WT-GrB. Afterward, an anti-His tag monoclonal antibody (diluted 1:5000) was applied at 37°C for 2 hours, followed by a 1.5-hour incubation with HRP-conjugated anti-mouse IgG (diluted 1:5000 in PBST) to detect bound antibodies. Finally, the chromogenic substrate TMB was added, and the reaction was allowed to develop for 15 minutes at 37°C. The color development was measured at 450 nm using an automated ELISA plate reader (Bio-Tek ELx800). To further evaluate the targeting ability of Z_HPV16E7-GrB in vitro, an indirect immunofluorescence assay was performed. SiHa, CaSki, HeLa, and C33A cells were seeded on glass slides and co-cultured with 100 μg/mL of Z_HPV16E7-GrB, Z_HPV16E7, or Z_WT-GrB for 24 hours. After incubation, the cells were fixed with 4% paraformaldehyde at room temperature for 10 minutes, followed by overnight blocking at 4°C in PBS containing 5% FBS. Subsequently, the slides were incubated overnight at 4°C with primary antibodies—either anti-GrB or anti-HPV16E7 (both diluted 1:1000). The following day, the samples were incubated for 1 hour at 37°C with secondary antibodies: FITC-conjugated goat anti-rabbit IgG (H+L) or Cy3-conjugated goat anti-mouse IgG (H+L) (both diluted 1:1000). Nuclei were counterstained with DAPI at 37°C for 5 minutes. Finally, fluorescence signals were analyzed using a Nikon fluorescence microscope (Japan).

To evaluate cell viability, 8,000 cells per well were seeded in 96-well plates. After a 24-hour adhesion period, cells were treated for 72 hours with either 100 μg/mL of Z_HPV16E7-GrB, Z_HPV16E7, or Z_WT-GrB, or 50 μg/mL oxaliplatin (positive control). Cell viability was then assessed using both the LDH Release Assay Kit and CellTiter-Lumi™ Steady-Glo Detection Kit according to the manufacturers’ protocols.

To investigate whether Z_HPV16E7-GrB could reverse EMT in cervical cancer cells, we performed a comprehensive analysis combining Transwell migration assays with EMT marker detection. First, we assessed cellular migration using Matrigel-coated Transwell inserts (8.0 µm pore size; Corning) in 24-well plates. After pretreating cells for 24 hours with 100 μg/mL of either Z_HPV16E7-GrB, Z_HPV16E7, or Z_WT-GrB, we seeded 1×10⁵ cells in serum-free medium into the upper chamber, while adding 600 μL of complete medium with 10% FBS to the lower chamber as a chemoattractant. Following 24-hour incubation, we carefully removed non-migrated cells from the upper membrane surface using PBS-moistened cotton swabs. The migrated cells on the lower surface were then fixed with 4% paraformaldehyde, stained with 0.4% crystal violet, and quantified by counting cells in five random 20× microscopic fields per insert. To directly examine EMT-related molecular changes, we performed Western blot analysis on cells treated identically for 24 hours. We probed for key EMT markers including epithelial markers (E-cadherin and N-cadherin), mesenchymal marker Vimentin, and transcription factor Snail.

To comprehensively assess Z_HPV16E7-GrB-induced apoptosis, we employed a multi-method approach combining TUNEL assay, flow cytometry, and Western blot analysis. Cervical cancer cells were treated with 100 μg/mL of Z_HPV16E7-GrB, Z_HPV16E7, or Z_WT-GrB for 18 hours before analysis. First, apoptosis was detected via the DeadEnd™ Fluorometric TUNEL System, with TUNEL-positive cells quantified under fluorescence microscopy. Second, treated cells were collected and resuspended at 3 × 10⁶ cells/mL for flow cytometry using Annexin V-FITC and propidium iodide staining on a FACSCanto II system (BD, USA), followed by data analysis with FlowJo V10.1. Third, Western blot analysis was performed to evaluate key apoptotic regulators, including pro-apoptotic Bax, anti-apoptotic Bcl-2, and the activated forms of caspase-8 and caspase-3, providing mechanistic insights into the apoptotic pathway triggered by Z_HPV16E7-GrB.

To systematically investigate Z_HPV16E7-GrB-induced pyroptosis, we employed an integrated analytical approach with the following experimental design: target cells were treated under standard culture conditions with 100 μg/mL of Z_HPV16E7-GrB, Z_HPV16E7, or Z_WT-GrB for 24 hours, followed by multiparametric evaluation. Initial assessment was performed using 1 μM SYTOX Green nucleic acid stain (37°C for 10 minutes) combined with fluorescence microscopy to quantify plasma membrane integrity loss as an indicator of early pyroptotic membrane rupture, while simultaneously measuring released inflammatory mediators (IL-1β, IL-18, and HMGB1) in supernatants using ELISA kits according to the manufacturer’s protocols. Subsequent microscopy analysis documented characteristic pyroptotic morphology including cellular swelling, membrane blebbing, and lysis, with Western blot analysis confirming GSDME cleavage as molecular evidence of pyroptosis. This comprehensive assessment strategy examined: (1) membrane integrity (SYTOX Green), (2) cytokine release (ELISA), (3) cellular morphology, and (4) molecular markers (GSDME cleavage).

To examine whether Z_HPV16E7-GrB-induced pyroptosis requires caspase-3 activity, we performed siRNA-mediated knockdown of caspase-3 in SiHa cells. Briefly, cells in 6-well plates were transfected with 1μg of caspase-3-specific siRNA (5’-CCGACAAGCUUGAAUUUAUTT-3’) or control nontargeting siRNA (5’-UUCUCCGAACGUGUCACGUTT-3’) using Lipofectamine™ 3000. 48 hours post-transfection, Western blot analysis was performed to verify the knockdown efficiency of caspase-3. These caspase-3-knockdown cells were then exposed to Z_HPV16E7-GrB (100 μg/mL) for 24 hours, after which pyroptosis was evaluated through multiple parameters: GSDME cleavage by Western blot analysis, membrane integrity by SYTOX Green uptake, inflammatory cytokine release via ELISA, - all performed according to our established pyroptosis assessment protocol.

To investigate whether Z_HPV16E7-GrB can directly cleave GSDME, we first established a eukaryotic expression system for FLAG-tagged GSDME. This was achieved by transfecting SiHa cells with the GSDME plasmid using Lipofectamine™ 3000 for 48 hours, followed by Western blot confirmation of successful expression. Following this, we purified the FLAG-GSDME protein from cell lysates using an anti-FLAG Affinity Gel kit, and verified the protein by both SDS-PAGE and Western blot analysis. Subsequently, to examine direct cleavage activity, we incubated 100 μg of the purified FLAG-GSDME with 100 μg of Z_HPV16E7-GrB, Z_HPV16E7, or Z_WT-GrB for 1 hour in reaction buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl), Western blot analysis was then performed to detect GSDME cleavage fragments.

Data are presented as means ± standard deviations (SD), unless otherwise specified. Statistical comparisons were exclusively performed within the same cell line. These comparisons were conducted using one-way ANOVA, followed by the least significant difference (LSD) test. A P value of < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS 22.0 software.

The optimized GrB (GrB(O)) sequence was designed through amino acid mutation (R201K) and substitution of nonpolar amino acids (R96A, R100A, R102A, K221A, K222A, K225A, R226A), as detailed in Table 1 . The corresponding nucleic acid sequence was synthesized and cloned into the pcDNA3.1 plasmid to create the pcDNA3.1/GrB(O) construct, as depicted in Figure 1A . SiHa cells were transfected with either the pcDNA3.1/GrB(O) plasmid or the pcDNA3.1/GrB(W) plasmid (wild-type GrB sequence). Post-transfection, GrB expression at the protein level was confirmed by Western blot analysis ( Figure 1B ). Immunofluorescence staining indicated the subcellular localization of GrB proteins ( Figure 1C ). Functional analyses revealed that both GrB(O) and GrB(W) induced apoptosis in transfected SiHa cells, as shown in Figures 1D, E . In addition to apoptosis, both constructs also promoted pyroptosis ( Figures 1F, G ), as evidenced by the cleavage of gasdermin E (GSDME), a key pyroptotic effector protein. This cleavage resulted in the generation of the active GSDME-N fragment ( Figures 1H, I ). This indicates that the optimized GrB retains its apoptotic and potential pyroptotic functionalities, as supported by the comparative analysis of both constructs.

Z_HPV16E7-GrB was successfully expressed in the E. coli protein production strain, yielding a protein with a molecular weight of approximately 36 kDa ( Figure 2A ). Western blot analysis confirmed the identity of the protein through recognition by both anti-His tag and anti-GrB antibodies ( Figure 2B ). Structural modeling revealed that the Z_HPV16E7 affibody, the flexible linker, and GrB form distinct, non-overlapping domains ( Figure 2C ), suggesting that the binding function of Z_HPV16E7-GrB is expected to be maintained. Binding assays demonstrated that Z_HPV16E7-GrB effectively binds to the HPV16E7 protein, confirming its targeted binding capability ( Figure 2D ). The binding titer of Z_HPV16E7-GrB to HPV16E7 was determined to be 1:3125 ( Figure 2E ). In contrast, Z_WT-GrB (an unscreened affibody conjugated to GrB) exhibited no binding activity. Immunofluorescence microscopy further validated the specificity of Z_HPV16E7-GrB. Colocalization with HPV16E7 was observed in HPV16-positive SiHa and CaSki cervical cancer cells ( Figure 2F ). No colocalization was detected in HPV18-positive HeLa cells (used as a type control) or HPV-negative C33A cells (used as a negative control). The study employed four representative cell lines: HPV16-positive SiHa and CaSki for target validation, HPV18-positive HeLa to evaluate potential cross-reactivity with the distinct HPV18 E7 oncoprotein (despite shared high-risk characteristics), and HPV-negative C33A as a fundamental baseline. This strategic selection enables rigorous demonstration of Z_HPV16E7-GrB’s exclusive specificity for HPV16 E7, as evidenced by: (1) differential responses between HPV16-positive versus HPV18-positive cells confirming E7-type discrimination, and (2) complete absence of activity in HPV-negative cells establishing target-dependence. These findings establish the specificity and high binding affinity of Z_HPV16E7-GrB toward HPV16E7. Collectively, these results highlight the potential of Z_HPV16E7-GrB as a targeted therapeutic agent, owing to its specificity, strong binding capability, and preserved functional domains.

The growth-inhibitory effects of Z_HPV16E7-GrB on HPV16-positive cells were evaluated through cell viability assays ( Figure 3A ) and lactate dehydrogenase (LDH) release, an indicator of cell damage ( Figure 3B ). Oxaliplatin, a clinically approved drug for advanced cervical cancer, was used as a positive control and demonstrated a significant reduction in cell viability alongside a marked increase in LDH release. Similarly, Z_HPV16E7-GrB substantially reduced cell viability and significantly increased LDH release compared to Z_HPV16E7 and Z_WT-GrB in both SiHa and CaSki cells. In contrast, no significant reduction in cell viability or increase in LDH release was observed with Z_HPV16E7-GrB in HeLa or C33A cells when compared to Z_HPV16E7 and Z_WT-GrB. These findings highlight the robust growth-inhibitory potential of Z_HPV16E7-GrB, underscoring its promise as a targeted therapeutic agent for HPV16-associated cervical cancer.

The E7 proteins of high-risk HPV types drive the malignant transformation of infected cells through various carcinogenic mechanisms, with cancer cell migration being a key malignant behavior mediated by E7. Existing literature establishes that HPV16 E6/E7 promotes EMT through the regulation of cadherins, specifically by downregulating E-cadherin and upregulating N-cadherin expression (18). To determine whether Z_HPV16E7-GrB could reverse this process by targeting E7, we assessed its impact on cell migration. The results showed that treatment with Z_HPV16E7-GrB or Z_HPV16E7 significantly inhibited cell migration, as evidenced by a reduced ability of SiHa and CaSki cells to cross the Matrigel layer, compared to equivalently treated HeLa and C33A cells ( Figures 4A, B ). Conversely, Z_WT-GrB, which lacks specific targeting, failed to inhibit migration in all tested cell lines.

Western blot analysis of EMT-related factors ( Figures 4C, D ) revealed that treatment of SiHa and CaSki cells with Z_HPV16E7-GrB or Z_HPV16E7 significantly altered the expression of key proteins governing cell migration and metastasis; specifically, both treatments caused a notable decrease in the mesenchymal regulators Vimentin and Snail (essential for EMT and associated with enhanced migration/metastasis) while significantly upregulating E-cadherin (a key protein maintaining intercellular adhesion and restricting migration); concurrently, expression of N-cadherin, another key cadherin involved in cell adhesion, was significantly decreased, resulting in a coordinated shift characterized by reduced N-cadherin and elevated E-cadherin – a reversal of the “cadherin switch” hallmark of EMT (26), where N-cadherin and E-cadherin play opposing yet interconnected roles in adhesion, tissue architecture, and metastasis; this dynamic shift, combined with the reduction in Vimentin and Snail, strongly indicates that Z_HPV16E7-GrB and Z_HPV16E7 inhibit the EMT process; in contrast, Z_WT-GrB-treated cells exhibited minimal changes in these factors, confirming that the observed EMT inhibition and cadherin expression reversal are direct consequences of HPV16 E7 targeting; collectively, these findings demonstrate that Z_HPV16E7 and Z_HPV16E7-GrB effectively inhibit the migration and invasion of HPV16-positive cervical cancer cells by E7-targeted disruption of EMT, likely through E7-targeted disruption of the EMT process.

GrB is an immune effector molecule that induces cell death via the apoptotic pathway. In this study, the ability of Z_HPV16E7-GrB to induce apoptosis in cervical cancer cells was assessed using TUNEL assays ( Figures 5A, B ) and flow cytometry ( Figures 5C, D , Annexin V/PI staining). The results demonstrated that Z_HPV16E7-GrB significantly induced apoptosis in HPV16-positive SiHa and CaSki cells, whereas no significant apoptosis was observed in the control HeLa and C33A cells.

Western blot analysis ( Figures 5E, F ) further validated these findings. In Z_HPV16E7-GrB-treated SiHa and CaSki cells, apoptosis was induced through the activation of caspase-3 and caspase-8. This was accompanied by a significant upregulation of the pro-apoptotic protein Bax, while the anti-apoptotic protein Bcl-2 was markedly downregulated. These results indicate that Z_HPV16E7-GrB effectively promotes apoptosis in HPV16-positive cervical cancer cells, highlighting its potential as a targeted therapeutic agent for HPV16-associated malignancies.

Recent studies have shown that in addition to inducing apoptosis, GrB can also trigger pyroptosis. To investigate whether Z_HPV16E7-GrB induces pyroptosis in HPV16-positive cervical cancer cells, we used SYTOX Green, a membrane-impermeable dye, to evaluate its effects. Z_HPV16E7-GrB treatment resulted in a significant increase in SYTOX Green fluorescence in SiHa and CaSki cells ( Figures 6A, B ), while no noticeable changes were observed in HeLa or C33A cells. Additionally, Z_HPV16E7-GrB treatment led to a marked release of inflammatory cytokines, including IL-18, IL-1β, and HMGB1, in SiHa and CaSki cells ( Figure 6C ), this release corresponded to the increase in SYTOX Green fluorescence, further confirming plasma membrane damage. In contrast, no significant release of inflammatory cytokines was detected in HeLa or C33A cells, confirming the HPV16-specificity of pyroptosis induction. We also assessed the key cytological features of pyroptosis in HPV16-positive cervical cancer cells. Under a light microscope, Z_HPV16E7-GrB-treated SiHa and CaSki cells exhibited distinct membrane bubbling ( Figure 6D , red arrows), a hallmark of pyroptosis.

Western blot analysis ( Figure 6E, F ) further validated these findings. Gasdermins, including GSDME, are critical mediators of pyroptosis. Upon cleavage, their N-terminal fragments form membrane pores, resulting in cell lysis and pyroptotic cell death. GSDME consists of an N-terminal (GSDME-N) and a C-terminal (GSDME-C) domain and primarily exists in its inactive full-length form (GSDME-F). Proteolytic cleavage of GSDME-F releases the active GSDME-N fragment, which integrates into the cell membrane, compromising its integrity. This process plays a pivotal role in tumor suppression and immune responses. Consistently, the cleaved GSDME-N fragment (35 kDa) was detected in SiHa and CaSki cells treated with Z_HPV16E7-GrB. These findings demonstrate that Z_HPV16E7-GrB effectively induces pyroptosis in HPV16-positive cervical cancer cells, unveiling a novel cell death mechanism with potential therapeutic applications.

Granzyme-induced pyroptosis is traditionally understood to occur through the caspase-3-dependent cleavage of GSDME. However, recent research suggests that GrB can also directly induce pyroptosis by cleaving GSDME in a caspase-3-independent manner. To investigate whether Z_HPV16E7-GrB cleaves GSDME via this alternative pathway, we conducted a caspase-3 interference experiment. Specific siRNAs targeting caspase-3 were transfected into SiHa cells, significantly suppressing caspase-3 expression, with siCaspase-3–3 showing the strongest inhibitory effect ( Figures 7A, B ), achieving over 93% knockdown efficiency.

Even with caspase-3 knockdown, cleaved GSDME-N fragments were still detected in Z_HPV16E7-GrB-treated SiHa cells, with band intensity comparable to that of the siNC control group ( Figures 7C, D ). Additionally, Z_HPV16E7-GrB continued to induce apoptosis, as evidenced by the SYTOX green fluorescence signals, which were similar to those observed in the siNC control group ( Figures 7E, F ). Moreover, the levels of released inflammatory cytokines IL-18, IL-1β, and HMGB1 were also comparable to those in the siNC control group ( Figure 7G ). These findings suggest that Z_HPV16E7-GrB can induce pyroptosis through the direct cleavage of GSDME, independent of caspase-3 activity.

Recent studies have demonstrated that GrB can induce pyroptosis through direct cleavage of GSDME (22). To investigate whether Z_HPV16E7-GrB utilizes this mechanism, we engineered a eukaryotic expression system for FLAG-tagged human GSDME. Following transient transfection in SiHa cells, FLAG-GSDME expression was confirmed by anti-FLAG Western blot ( Figure 8A ), with subsequent purification via anti-FLAG affinity chromatography yielding a predominant 55-kDa band corresponding to full-length GSDME ( Figure 8B ), which was specifically recognized by anti-GSDME antibodies ( Figure 8C ).

In vitro cleavage assays revealed that both Z_HPV16E7-GrB and Z_WT-GrB—but not the affibody control Z_HPV16E7—efficiently cleaved GSDME ( Figure 8D ), demonstrating that Z_HPV16E7-GrB induces pyroptosis in HPV16-positive cells through direct cleavage of GSDME by GrB.